DEVELOPMENTAL DYNAMICS 214:323–336 (1999) Early Hematopoiesis and Developing Lymphoid Organs in the Zebrafish CATHERINE E. WILLETT,1* ALFONSO CORTES,2 ADELINA ZUASTI,3 AND AGUSTIN G. ZAPATA2 Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 2Department of Cell Biology, Faculty of Biology, Complutense University, Madrid, Spain 3Department of Cell Biology, School of Medicine, University of Murcia, Murcia, Spain 1Biology ABSTRACT In zebrafish, the transparent and rapidly developing embryo and the potential for genetic screening offer a unique opportunity to investigate the early development of the vertebrate immune system. Here we describe the initial appearance of various blood lineages and the nature of accumulating hematopoietic tissue in the thymus and kidney, the main lymphoid organs of adult teleosts. The ultrastructure of the first site of hematopoiesis, the intermediate cell mass (ICM), is described from the 5-somite stage, about 11.5 hours post-fertilization (hpf) until 24 hpf. The ICM gives rise to the primitive erythroid lineage, which accounts for all circulating erythrocytes for the first 4 days pf. From 24 to 72 hpf, a few developing granulocytes are seen close to the yolk sac walls and in the caudal axial vein. The heart, previously proposed as an early bloodforming organ in zebrafish, did not contain hematopoietic cells. The thymic primordium, consisting of two layers of epithelial cells, appears at 60 hpf. By 65 hpf, it is colonized by immature lymphoblasts. The thymus gradually accumulates lymphocytes, and the lymphocytes and epithelial cells progressively differentiate for 3 weeks pf. At 96 hr, the pronephros contains hematopoietic cells, mainly developing erythrocytes and granulocytes. The amount of renal hematopoietic tissue increases rapidly; however, lymphocytes were not detected until 3 weeks pf. Dev Dyn 1999;214:323–336. r 1999 Wiley-Liss, Inc. Key words: zebrafish development; hematopoiesis; thymus; kidney INTRODUCTION In adult vertebrates, erythroid, myeloid, and lymphoid cells share a common progenitor. It is thought that these common stem cells gives rise to two subsequent progenitors, one capable of generating erythroid and myeloid lineages, the other generating lymphoid lineages. The myeloid lineage includes monocytes (macrophages) and granulocytes. Hematopoietic tissue can therefore be erythropoietic, myelopoietic, lymphopoietic, or a combination, depending on the nature of the progenitor cells comprising it. r 1999 WILEY-LISS, INC. In all vertebrates, the site of hematopoiesis changes during development. In mammals, chicken, and Xenopus laevis, the first site of hematopoiesis is in the tissue derived from the ventral mesoderm surrounding the yolk sac—the primitive blood islands in mammals and chicken, and the ventral blood islands of Xenopus (reviewed in Orkin, 1995; Robb, 1997). This stage of hematopoiesis is called ‘‘primitive,’’ and this tissue gives rise only to embryonic red blood cells (RBCs) and some myeloid lineages. The next site of hematopoiesis differs among species; however, the origin of the tissue in all cases is dorsal mesoderm: the para-aortic splanchopleura and the related aorta-gonad-mesonephros region (AGM) in mammals (Godin et al., 1995; Medvinsky and Dzierzak, 1996) and chicken (Dieterlen-Lievre and Le Douarin, 1993); the dorso-lateral plate (DLP) in Xenopus (Chen and Turpen, 1995). This dorsallyderived hematopoietic tissue is capable of giving rise to additional blood lineages, including lymphoid (Cumano et al., 1996; Medvinsky and Dzierzak, 1996), and is called ‘‘definitive’’ hematopoiesis. Sites of hematopoiesis during the development of several teleost species have been described (Al-Adhami and Kunz, 1976; Al-Adhami and Kunz, 1977; Ellis, 1977; Iuchi and Yamamoto, 1983; Secombes et al., 1983; Zapata et al., 1995). There are three classes of teleost embryos, based on the initial site of hematopoiesis. In some species, such as angelfish (Al-Adhami and Kunz, 1976; Colle-Vandevelde, 1962) and Blennius gattorugine (Colle-Vandevelde, 1963), initial hematopoiesis occurs exclusively in the yolk sac blood islands. In other species such as zebrafish, the first and only hematopoietic site during embryogenesis is the intraembryonic ICM (Al-Adhami and Kunz, 1977; Glomski et al., 1992; this communication). In yet other species, such as killifish (Stockard, 1915a; Stockard, 1915b), rainbow trout (Iuchi and Yamamoto, 1983), and Lebistes reticulatus (Colle-Vandevelde, 1961a; Colle-Vandevelde, 1961b) early hematopoiesis occurs at both locations. Analogous Grant sponsor: National Institutes of Health; Grant number: 2 RO1 AI08054; Grant sponsor: Spanish Ministry of Education and Culture; Grant number: PB94–0332. *Correspondence to: Catherine E. Willett, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: firstname.lastname@example.org Received 5 November 1998; Accepted 23 December 1998 324 WILLETT ET AL. to yolk sac hematopoiesis of other vertebrates, both the yolk sac blood islands and ICM of teleosts, which are derived from ventral lateral mesoderm, are thought to be sites of primitive hematopoiesis. Recent reports in zebrafish, describing expression patterns of transcription factors known to be involved in murine hematopoiesis, support the idea that the ICM is the initial site of blood formation and gives rise primarily to primitive erythrocytes (Detrich et al., 1995; Liao et al., 1998; Thompson et al., 1998). In zebrafish, cells comprising the ICM enter the developing circulatory system between 24 and 30 hr post-fertilization (hpf). Recent expression studies of transcription factors involved in hematopoiesis suggest that a few pluripotent hematopoietic cells subsequently exist at other locations, most notably the ventral wall of the dorsal aorta and a region in the tail posterior to the ICM and ventral to the axial vein, the ‘‘ventral vein region’’ (Liao et al., 1998; Thompson et al., 1998; H. Kawasaki and C. Amemiya, personal communication; our unpublished results). These putative hematopoietic foci may constitute the initial site of definitive hematopoiesis in zebrafish. Later, hematopoietic tissue is seen in the kidney (Al-Adhami and Kunz, 1977; Willett et al., 1997b). The teleost kidney is the equivalent of bone marrow in other vertebrates and is the major site of hematopoiesis through adulthood (reviewed in Zapata et al., 1995). The zebrafish has recently shown promise as a model organism for the study of vertebrate embryogenesis— the transparent, free-living, and rapidly developing embryos are ideal for observation of early developmental processes (Kimmel, 1989). An additional advantage of zebrafish as a model system is the ability to perform large-scale screens for mutations affecting embryogenesis (Driever et al., 1996; Hafter et al., 1996). To initiate analysis of the zebrafish immune system, we cloned the zebrafish rag1 and rag2 genes, which encode the recombinase responsible for recombination of the Ig and TCR genes. Expression of rag1 identified the thymus as the first lymphoid organ to emerge during zebrafish ontogeny (Willett et al., 1997a; Willett et al., 1997b). A previous histological description performed by light microscopy had identified primitive hematopoietic cells in the ICM at 10 somites; hematopoietic cells were first described in the pronephros on day 4 pf, with ‘‘all lineages’’ visible by day 8 pf (Al-Adhami and Kunz, 1977). It was not clear, however, whether they included lymphocytes in the description, and the thymus was not mentioned. In our initial studies by light microscopy, we described isolated cells of possible hematopoietic nature in the pronephros at 90 hpf, although significant hematopoietic tissue did not accumulate until 2–3 weeks pf (Willett et al., 1997b). In contrast, accumulation of lymphoid cells in the thymus occurred by 72 hpf, supporting the observation that, in most teleosts, the thymus is the first organ to become lymphoid (Zapata et al., 1997). A detailed description of the development of the zebrafish immune system is required for phenotypic characterization of any mutants that might be isolated in future screens. The following specific points are addressed here: (1) the timing of lymphocyte infiltration and maturation within the thymus, (2) the timing of differentiation of the epithelial components of the thymus, (3) the possible presence of hematopoietic tissue in the heart, and (4) the discrepancy between our observations and the report of Al-Adhami and Kunz regarding the timing of appearance of hematopoietic lineages in the pronephros. We have thus extended our previous results and present a detailed histological description by light and electron microscopy of the zebrafish immune system from 5 somites through 3 weeks of development. RESULTS Initial Site of Hematopoiesis As early as 11 hr post-fertilization (the 5-somite stage), cells can be identified in an intraembryonic region between the somites and the yolk sac, which presumably corresponds to the intermediate cell mass (ICM) described by other authors (Al-Adhami and Kunz, 1977; Detrich et al., 1995; Fig. 1A,B). This area contains primitive hematopoietic precursors (Fig. 1B) in addition to cells that will give rise to the pronephros and trunk blood vessels. By 20 hpf, these cells have migrated medially and have aggregated into two bilaterally symmetric bands between the notochord and endoderm (Fig. 1C), as previously noted (Detrich et al., 1995). The 20 hpf ICM contains proerythroblasts, which exhibit an electron-dense nucleus with clumps of condensed chromatin and a cytoplasm filled by ribosomes and large, electron-lucent mitochondria (Fig. 1D). These cells also contain ropheocytic vesicles, which are involved in iron transport, and are a hallmark of erythroid cells. Myeloblasts are not evident. At 24 hpf, numerous erythroblasts appear, closely associated with primitive endothelial cells proximal to the developing pronephric ducts (Fig. 2A). Erythroblasts, round or polygonal in shape, possess clumps of condensed chromatin arranged in an electron-dense nuclear matrix. The cytoplasm contains ropheocytic vesicles and is filled with polyribosomes (Fig. 2B). Between 24 and 30 hpf, the ICM disappears as cells are taken up by the nascent circulatory system. At 42 hpf, a few cells that morphologically resemble erythroblasts and promonocytes were identified in the ventral axial vein region of the tail, posterior to the ICM (Fig. 2C,D). By electron microscopy (EM), there are circulating cells in the axial vein (Fig. 2C); however, there were also some primitive cells in the connective tissue surrounding the blood vessel (Fig. 2D). Early Myelopoiesis Although not seen in the ICM, cells of the myeloid lineage can be found surrounding the yolk sac as early as 24 hpf (data not shown). Fig. 3 shows myeloblasts HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT 325 Fig. 1. First appearance of hematopoietic tissue in the intermediate cell mass (ICM). A, B: Transverse section of 11 hpf embryo (5 somites). C, D: 20 hpf embryo. A: The forming ICM at 5 somites (circled) contains precursors that will give rise to the first hematopoietic cells, the pronephros, and blood vessels. NT, neural tube; Nc, notochord; S, somite; YS, yolk sac. Light micrograph, magnification: 120 ⫻. B: Primitive hematopoietic cell in forming ICM. arrows, polyribosomes; n, nucleus; m, mitochondria. Electron micrograph, magnification: 7,800 ⫻. C: ICM (circled) is dorsal and medial to the pronephric ducts (PD). Note the abundance of erythroblasts (arrows). S, somite; YS, yolk sac. Light micrograph, magnification: 250 ⫻. D: Erythroblasts (Eb) can be seen in the ICM adjacent to the pronephric duct (PD). Note the high electron density of the nucleus (n) and the abundance of ribosomes and large mitochondria (m) in the cytoplasm of erythroblasts. Primitive erythroblasts contain ropheocytic vesicles (arrow). Electron micrograph, magnification: 5,900 ⫻. Plane of sections diagrammed at right. and neutrophilic myelocytes at 34 and 48 hpf. Whereas numerous circulating erythroblasts occur inside blood vessels, maturing granulocytes are seen mostly outside the circulatory system, in loose connective tissue surrounding the yolk sac at 34 hpf (Fig. 3A), and adjacent to the first renal tubules at 48 hpf (Fig. 3B), although some images support the migration of myelocytes or young neutrophils through the blood vessel walls (Fig. 3C). Mature neutrophils can be identified in different tissues at 72 hpf (data not shown). resembling lymphocytes or primitive hematopoietic cells similar to those described by Al-Adhami and Kunz (1977) seemed to occur in the endocardial walls (Fig. 4E). However, at higher magnifications by EM, these cells were found to be irregular endocardial cells joined together by desmosomes, specialized epithelial cell junctions (Fig. 4F). Circulating Erythroblasts in the Heart A previous description of hematopoiesis in zebrafish by light microscopy suggested that the zebrafish heart functions as a transitory blood-forming organ, between the time of disappearance of the ICM and the onset of hematopoiesis in the kidney (Al-Adhami and Kunz, 1977). We therefore examined the histology of the heart from 24 hpf onward. In all stages examined, circulating erythroblasts could easily be identified (Fig. 4A,B,C), sometimes closely associated with the endocardium (Fig. 4A,C,D). At lower magnifications, small cells The Thymus We previously reported the appearance of the thymic primordium by light microscopy at 54 hpf (Willett et al., 1997b). Figs. 5A,B show the primordium at 60 hpf as a small outgrowth of the pharyngeal epithelium, devoid of lymphocytes. By EM, the region containing the primordium can be seen to consist of two layers of epithelial cells surrounded by an incipient but continuous basement membrane (Fig. 5B). Flat, electron-dense epithelial cells line the pharyngeal cavity, joined together by well-developed desmosomes. Adjacent to the pharyngeal epithelium, electron-lucent epithelial cells constitute the thymic primordium. These epithelial cells are irregular or polygonal, contain numerous 326 WILLETT ET AL. Fig. 2. ICM at 24 hpf (A, B) and ventral vein region of tail at 42 hpf (C, D); transverse sections. A: Erythroblasts (Eb) are closely associated with primitive endothelial cells (En) adjacent to the pronephric duct (PD). Electron micrograph, magnification: 2,600 ⫻. The box in (A) is magnified in (B). Both the nucleus (n) and cytoplasm of erythroblasts are electrondense. The cytoplasm has abundant ribosomes, electron-lucent, round mitochondria (m), and occasional ropheocytic vesicles (arrows). Electron micrograph, magnification: 8,500 ⫻. C: Erythroblasts (Eb) and presump- tive promonocyte (arrow) in lumen of axial vein in tail. Electron micrograph, magnification: 3,300 ⫻. D: Primitive hematopoietic cell, presumably a promonocyte or early promyelocyte (Pm), in connective tissue adjacent to axial vein, surrounded by fibroblasts (Fb). Two lobes of the nucleus (n) can be seen, along with numerous mitochondria (m) and rough endoplasmic reticulum (r). Electron micrograph, magnification: 5,500 ⫻. ribosomes, large electron-lucent mitochondria, and rough endoplasmic reticulum. Nascent desmosomes join these primitive epithelial cells together (Fig. 5C). At 65 hpf, the thymic primordium is slightly larger, and by EM, the first lymphoid cells can be seen (Fig. 5D,E). The thymic epithelial cells arranged along the pharyngeal epithelium are relatively primitive, and the basement membrane is still poorly formed. Lymphoblasts can be distinguished, between epithelial cells, by their round shape, high electron density, and a small amount of cytoplasm filled with ribosomes, yet missing rough endoplasmic reticulum. The thymus grows slowly for the next few hours, and the number of lymphoblasts increases gradually. At 72 hpf, the thymic primordium still contains few lymphoid cells. However, the connections between epithelial cells have matured (data not shown). Thymic epithelial cells now contain an irregular nucleus with few peripheral areas of condensed chromatin (data not shown). The thymus contains many more lymphoid cells at 96 hpf (Fig. 6A). Lymphoblasts are seen as large cells with a nucleus containing few areas of condensed chromatin, a prominent nucleolus, numerous polyribosomes, a poorly developed Golgi complex, and some cisternae of rough endoplasmic reticulum (Fig 6B). The cytoplasm of the thymic epithelial cells contain bundles of microfilaments, and frequent desmosomes occur between these epithelial cells, two characteristic features of maturing epithelial cells (Fig. 6C). On day 7 pf, increased numbers of small (mature) lymphocytes, as well as additional types of epithelial cells, are found in the thymus (Fig. 6D), as is seen in the HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT 327 Fig. 3. First appearance of myeloid lineages. A: 34 hpf embryo. Myeloid cells are found outside of the blood vessel (BV), both close to the yolk sac (YS), and next to connective tissue (CT) of the body wall. arrows, myeloblasts; arrowheads, neutrophilic promyelocytes. Electron micrograph, magnification: 2,300 ⫻. B: 48 hpf embryo. Neutrophilic myelocyte (see arrows in box) in the connective tissue surrounding the ciliated pronephric duct (PD), between the yolk sac (YS), and yolk sac blood vessel (BV; Duct of Cuvier). Electron micrograph, magnification: 2,900 ⫻. C: Higher magnification of the neutrophilic myelocyte boxed in (B). Note the immature cytoplasmic granules and the Golgi complex (g). Cell processes (arrows) crossing two endothelial cells (En) suggest that the cell is migrating into the blood circulation. Electron micrograph, magnification: 10,200 ⫻. Plane of section and locations of micrographs diagrammed at upper right. G, glomerulus; I, intestine; n, nucleus; Nc, notochord; Nt, neural tube; PD, pronephric duct; S, somite; YS, yolk sac. trout thymus (Castillo et al., 1991). Numerous medium and small lymphocytes are seen as round, electrondense cells with abundant clumps of condensed chromatin and a small cytoplasm filled with ribosomes. Electron-dense epithelial cells are seen between the lymphoid cells. Secretory epithelial cells containing a heterogeneous population of electron-dense, cytoplas- mic granules and a well developed Golgi complex are now found (Fig. 6E). By 3 weeks pf, both electron lucent epithelial cells (Fig. 7A) and epithelial cysts (Fig. 7B) are seen, as has been reported in the thymus of other fish (Zapata et al., 1996), marking the morphological maturation of the thymic stroma which began 1 week pf. The pale epithe- 328 WILLETT ET AL. Fig. 4. Circulating erythroblasts in heart. A: Transverse section through 24 hpf heart showing erythroblasts (arrows) closely associated with endocardial cells (Ec). Light micrograph, magnification 1,000 ⫻. B: Transverse section through 60 hpf heart showing immature erythrocytes (RBC) within the heart chambers. Ec, endocardial cells. Light micrograph, magnification: 800 ⫻. C: Transverse section through 72 hpf heart showing pooled erythrocytes and erythroblasts in atrium and ventricle. GA, gill arches; H, heart; Ph, pharynx. Inset: magnification of boxed region. Circulating erythroblasts (arrows) are evident. Light micrograph, magnification: 170 ⫻; inset, 600 ⫻. D: Circulating erythroblasts (Eb) in close association with the endocardium (Ec) at 72 hpf. Mc, myocardium. Electron micrograph, magnification: 3,400 ⫻. E: Lymphoid-like cells in endocardium (arrows) at 72 hpf. Electron micrograph, magnification: 3,400 ⫻. Boxed area in (E) is magnified in (F). F: Lymphoid-like cells are actually irregular endocardial cells connected together by desmosomes, specialized cell junctions (arrows). Electron micrograph, magnification: 13,200 ⫻. lial cells have the same general features as the epithelial cells described above at 72 hpf and 96 hpf, but they have lost electron-dense cytoplasm and appear devoid of most membranous organelles. They contain bundles of cytoplasmic filaments (Fig. 7C) and are connected by desmosomes to the neighboring electron-dense epithelial cells (Fig. 7D). The Kidney Well-developed renal tubules and glomeruli are observed at 72 hpf; however, the first hematopoietic cells do not appear in the zebrafish pronephros until 96 hpf (Fig. 8A,B). Hematopoietic tissue is organized into cell cords surrounding blood vessels, between the renal tubules and glomeruli. By day 7 pf, fibroblastic reticular cells line the sinusoidal blood vessels; this constitutes the supporting apparatus for the developing hematopoietic cells (Fig. 8C,D), as occurs in the kidney of other teleosts (Meseguer et al., 1991; Zapata, 1979). Fibroblastic reticular cells and cells lining the sinusoidal blood vessels are both electron-lucent, the former being very irregular and extremely flat, the latter HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT 329 Fig. 5. Thymic primordium 60 hpf (A, B, C) and 65 hpf (D, E). A: Thymic primordium (boxed) lies in a dorsal pocket of the pharynx, ventral to the otic vesicle (Ov). Ph, pharyngeal epithelium. Light micrograph, magnification: 225 ⫻. B: Boxed area in (A), magnified. Thymic primordium consists of two epithelial cell layers, the pharyngeal epithelium (Ph), and thymic epithelium (TE). Pharyngeal epithelial cells are joined by desmosomes (arrow). Limit of thymic primordium indicated by arrowheads. m, large mitochondria; r, rough endoplasmic reticulum. Electron micrograph, magnification: 9,400 ⫻. C: Incipient desmosomes (arrows) between thymic epithelial cells. Electron micrograph, magnification: 54,500 ⫻. D: By 65 hpf, lymphoblasts (Lb) have infiltrated between epithelial cells (TE). Ph, pharyngeal epithelium. Electron micrograph, magnification: 4,100 ⫻. E: Lymphoblasts are distinguished by patches of condensed chromatin. Thymic epithelial cells have prominent mitochondria (m) and rough endoplasmic reticulum (r). TE, epithelial cells. Arrows mark basement membrane. Electron micrograph, magnification: 12,200 ⫻. exhibiting an elongated nucleus with peripheral areas of condensed chromatin, and sometimes, a small, compact nucleolus (Fig. 8D). It was difficult to observe a basement membrane separating these cell types. At these early stages, the renal hematopoietic tissue consists of erythroblasts, myeloblasts, and heterophilic myelocytes, which form clusters or cords of cells (Fig. 8C). During the next several days, the amount of hematopoietic tissue greatly increases (Fig. 9A). However, even at 2 weeks, the types of cells found in the pronephros remain remarkably unchanged; erythroblasts and myeloblasts are still the most frequent blood cell lineages found (Fig. 9B,C). Erythroblasts, at different stages of development, are easily identifiable by the high electron density of both their nuclei and cytoplasm (Fig. 9B). Characteristic of erythroblasts is a nucleus with many patches of condensed chromatin and a small nucleolus. In addition, hemoglobin, which leaks from the cytoplasm, accumulates in the nuclear matrix and confers high electron density to the nucleus, which distinguishes these cells from myeloblasts or lymphoblasts. The hemoglobin-filled cytoplasm contains large, electron-lucent vacuoles, as a consequence of the degradation of membranous organelles (i.e., mitochondria, lysosomes). Macrophages can be seen with degenerated erythroid cells in their cytoplasm (Fig. 9B). Myeloblasts can be seen as polygonal cells, larger than erythroblasts or lymphoblasts, with few patches of condensed chromatin, an active, big nucleolus, and a cytoplasm filled with abundant ribosomes and a few electron-lucent mitochondria (Fig. 9C). All developmental stages of the neutrophilic cell lineage could be identified at 2 weeks pf. At 3 weeks pf, the pronephros has enlarged and contains more patches of hematopoietic tissue, interspersed among increased numbers of renal tubules (Fig. 9D). The first lymphoid cells are seen in the pronephros at this time (Fig. 9E,9F). Lymphoblasts are identified as being smaller than myeloblasts, with a small cytoplasm filled by ribosomes and a few clumps of 330 WILLETT ET AL. Fig. 6. Thymic primordium 96 hpf (A,B,C) and day 7 pf (D,E). A: Thymus (circled) contains many lymphoid cells. Ph, pharyngeal epithelium. Light micrograph, magnification: 630 ⫻. B: Lymphoblasts contain a nucleus (n) with clumps of condensed chromatin, a nucleolus (nu), and cytoplasm filled with abundant ribosomes. m, mitochondria; g, Golgi complex; r, cisternae of rough endoplasmic reticulum. Electron micrograph, magnification: 11,100 ⫻. C: Filaments (arrows) in, and desmosomes (arrowheads) between, two thymic epithelial cell processes. Electron micrograph, magnification: 21,600 ⫻. D: Increasing numbers of medium and small lymphocytes (arrowheads) occur among thymic epithelial cells (stars). A connective tissue capsule (demarcated by arrows) surrounds the thymus. Ph, pharyngeal epithelium. Electron micrograph, magnification: 4,100 ⫻. E: Thymic epithelial cells have begun to differentiate into different cell types, including secretory epithelial cell shown here. stars, basement membrane; arrowheads, desmosomes; arrows, secretory granules. Electron micrograph, magnification: 13,500 ⫻. condensed chromatin (Fig. 9E). Small lymphocytes have very electron-dense cytoplasm, filled by free ribosomes and devoid of membranous organelles, which forms a narrow rim around a large nucleus that has abundant patches of condensed chromatin (Fig. 9F). Hematopoietic cells in ICM are evident at the 5-somite stage (10–11 hpf; Fig. 1A,B). The morphological features of these early progenitor cells are not sufficiently defined to distinguish hematopoietic tissue from vascular and pronephric progenitors, which also originate in this region (Gering et al., 1998; Liao et al., 1998; Liao et al., 1997). By 20 hpf, proerythroblasts are clearly distinguished (Fig. 1C,D). Accumulation of hematopoietic tissue in the ICM reaches its maximum development at 16 to 24 hpf. Erythroblasts within the ICM are taken up into the nascent circulatory system around 24 hpf and begin to circulate as immature erythrocytes (Fig. 2; Al-Adhami and Kunz, 1977; Detrich et al., 1995; Weinstein et al., 1996); final maturation of these erythrocytes occurs in circulation. These results suggest that the ICM is responsible for ‘‘primitive’’ hematopoiesis and is functionally analogous to the yolk sac blood islands of mammals and birds and the ventral blood islands of amphibians. DISCUSSION Recent results in zebrafish have demonstrated that the ICM is the first embryonic area expressing molecules involved in early blood cell formation in mammals, including scl, GATA-1, GATA-2, c-myb, LMO2 and Ikaros (Detrich et al., 1995; Gering et al., 1998; Liao et al., 1998; Thompson et al., 1998; H. Kawasaki and C. Amemiya, personal communication; unpublished results of the authors). The observations presented here, by light and electron microscopy, confirm those studies and show that the ICM contains hematopoeitic/erythropoietic progenitor cells. HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT 331 Fig. 7. Different types of epithelial cells are seen in 3 week pf thymus. A: Electron-lucent (pale) epithelial cell (star), surrounded by lymphoid cells. Electron micrograph, magnification: 8,800 ⫻. B: An epithelial cyst. Epithelial cell processes, which delimit the cavity of the epithelial cyst, are marked by arrows. Electron micrograph, magnification: 8,900 ⫻. C: Bundles of microfilaments (arrows) in the cytoplasm of an electronlucent thymic epithelial cell. Electron micrograph, magnification: 48,800 ⫻. D: Desmosomes connect cell processes of an electron-dense (small stars) and an electron-lucent thymic epithelial cell (star). Electron micrograph, magnification: 48,800 ⫻. The location of hematopoietic tissue, from the time the ICM or yolk sac blood islands disappear (24–30 hpf in zebrafish) until the appearance of hematopoietic tissue in the kidney (at 96 hpf in zebrafish; Fig 8), has not been conclusively identified in teleosts. Thompson et al. (1998) suggest that a lateral stripe of c-Mybexpressing cells may populate the ventral wall of the dorsal aorta at 48 hpf. The dorsal aorta may therefore represent the teleost equivalent of the aorta-gonadmesonephros region (AGM), which has been shown to be the first site of definitive hematopoiesis in Xenopus (Chen and Turpen, 1995), chicken (Dieterlen-Lievre and Le Douarin, 1993), and mouse (Godin et al., 1995; Medvinsky and Dzierzak, 1996), and has also been described in elasmobranchs (Zapata et al., 1996). Definitive resolution of the identity of these cells awaits lineage tracing experiments. Liao et al. (1998) and Thompson et al. (1998) report expression of hematopoietic markers that persists until day 4 pf in the region of the tail posterior to the ICM and ventral to the axial vein. At 42 hpf, a few erythro- blasts and promonocytes are found at this location (Fig. 2C,2D); these cells are found in the axial vein as well as in the mesenchyme surrounding the blood vessel. This population of cells could represent a residual compartment of the ICM that might be capable of generating hematopoietic precursors for a longer period during embryogenesis than previously thought. Between 24 and 48 hpf, a few myeloblasts and heterophilic myelocytes appear between the yolk sac and the body walls (data not shown; see Fig. 3).Mature neutrophils are seen in different tissues, but not in the blood circulation, at 72 hpf. Detrich et al (1995) reported the migration of erythroblasts from the ICM onto the yolk sac between the ectoderm and the yolk surface, the same location in which the myeloid cells occur. Macrophages have also been reported between the yolk sac and body walls, by light microscopy, in living embryos from 24 hpf (P. Herbomel, personal communication). Al-Adhami and Kunz (1976, 1977) postulated the existence of endocardial ‘‘stem-cell like’’ cells in both 332 WILLETT ET AL. Fig. 8. Pronephros of 96 hpf (A, B) and day 7 pf (C, D) embryo. A: Hematopoietic tissue between renal tubules (RT) and the forming glomerulus (G). BV, blood vessel; I, intestine; L, liver; M, muscle; Nc, notochord. Dorsal is upper right corner. Transverse section, light micrograph, magnification: 180 ⫻. B: Magnification of boxed area in (A). Mature (arrows) and developing (white arrowhead) erythrocytes can be seen. G, glomerulus. Light micrograph, magnification: 540 ⫻. C: Hematopoietic cell cords are arranged between blood vessels (BV) and sinusoidal blood vessels (SBV) and consist of myeloid (MC) and erythroid cells (E). In the lumen of a sinusoidal blood vessel, erythroblasts (Eb) are closely associated with cells lining the lumen (stars). arrowheads, Fibroblastic reticular cells. Electron micrograph, magnification: 3,700 ⫻. D: Box in (C), magnified. Erythroblast (Eb) associated with fibroblastic reticular cells (arrowheads) and cells lining the lumen (stars) of a sinusoidal blood vessel. Note the similarities between the reticular cell and the lining cell, and the absence of a basement membrane separating them. Electron micrograph, magnification: 8,300 ⫻. zebrafish and angelfish that could become free hematopoietic stem cells. This suggests that the heart could be the blood cell forming organ, between the time of disappearance of hematopoiesis in the ICM and the onset of blood cell formation in the pronephros. Our analyses, by both light and electron microscopy, demonstrate the existence of primitive hematopoietic cells, mainly erythroblasts, closely associated with, but not attached to, the endocardial cells of zebrafish heart from 24 hpf onward (Fig. 4). These cells are circulating erythroblasts and immature erythrocytes, which, as mentioned above, mature in circulation (Fig. 4A,B,C,D). There are also immature cells in the endocardium itself (Fig. 4E). However, upon inspection by EM, these cells are found to be connected by desmosomes and are, in fact, maturing endocardial cells (Fig. 4F). A similar situation has recently been described in the developing heart of the turbot (Padrós and Crespo, 1996). Liao et al. (1998) observed scl-expressing cells in the heart at day 4 pf; however, upon sectioning, these cells were shown to be pooled circulating blood cells. Maturation of lymphocytes in the thymus is dependent on interactions with the thymic epithelium; interestingly, the converse is also true (Hollander et al., 1995; Shores et al., 1991; van Vleit et al., 1985). In teleosts, several epithelial cell types have been described (reviewed in Chilmonczyk, 1992). At the time of initial infiltration by lymphoblasts, the thymic epithelial cells are relatively undifferentiated. Differentiation of the various epithelial cell types continues long after infiltration (Castillo et al., 1991). While the function of different classes of thymic epithelial cells has not been addressed in teleosts, certain morphological similarities to classes of mammalian cell types have been noted (Castillo et al., 1991). In zebrafish, the thymic epithelium is first visible, by light and electron microscopy, at 60 hpf, lining the HEMATOPOIESIS DURING ZEBRAFISH DEVELOPMENT 333 Fig. 9. Pronephros at 2 weeks (A, B, C) and 3 weeks (D, E, F) pf. A: Hematopoietic tissue (circled) surrounding the renal tubules (RT) has expanded. G, glomerulus; I, intestine; L, liver; M, muscle; Nc, notochord. Light micrograph, magnification: 110 ⫻. B: Macrophage (Mp) containing engulfed cell debris appears between erythroblasts. Note the high electron density of both nuclei and cytoplasm of the erythroblasts and the presence of big vacuoles (arrowheads), which represent degenerated membranous organelles. Electron micrograph, magnification: 3,900 ⫻. C: Renal hematopoietic tissue also contains myeloblasts (Mb), myelocytes (Mc) and mature neutrophils (Ne). Myeloblasts are large cells with few areas of condensed chromatin, an obvious nucleolus (nu), abundant ribosomes and incipient rough endoplasmic reticulum (arrowheads). Neutrophils contain abundant cytoplasmic granules (gr). arrow, Fibroblastic reticular cell; m, mitochondria. Electron micrograph, magnification: 5,400 ⫻. D: Pronephros has enlarged and contains increased hematopoietic tissue (circled) interspersed among renal tubules (RT) and blood vessels (BV). Light micrograph, magnification: 100 ⫻. E: Several types of hematopoietic cells are discernible, including small (SL), medium (ML), and large (LL) lymphocytes, myeloblasts (Mb), mature neutrophils (Ne), and mature acidophils (Ac). Electron micrograph, magnification: 3,200 ⫻. F: Small lymphocytes (SL) exhibit high electron density, numerous clumps of condensed chromatin, and a small rim of cytoplasm packed with ribosomes. Electron micrograph, magnification: 12,300 ⫻. dorsal pharynx (Fig. 5A,B,C); the thymic epithelium is continuous with the pharyngeal epithelium. Lymphoblasts can first be distinguished in the thymic primordium by EM at 65 hpf (Fig. 5D,E). The epithelial cells are still relatively immature. At 72 hpf, the number of lymphoid cells has increased slightly, and the epithelial cells show signs of maturation; some are developing secretory capability, desmosomes are forming between cells, and a basement membrane is forming. The hematopoietic transcription factor Ikaros can be detected in a few of these thymic lymphocytes at 72 hpf (H. Kawasaki and C. Amemiya, personal communication; unpublished results of the authors). By 92 hpf, the thymus has enlarged considerably and contains many more lymphoid cells (Fig. 6). At this approximate time, rag1 expression is first seen in the thymus (Willett et al., 1997b). Lymphoblasts remain the most numerous lymphoid cell type, with increasing numbers of medium and small lymphocytes during the first week pf. Epithe- lial cells have mature desmosomes connecting them, and the cytoplasm has developed bundles of microfilaments. From day 7 pf through 3 weeks pf, increasing heterogeneity is seen in both the lymphocytic and epithelial cell populations (Fig. 6C,D, and Fig. 7). The sequential expression of Ikaros and rag1, as well as increased heterogeneity, suggest progressive maturation of lymphocytes in the thymic primordium, which correlates with the progressive maturation of thymic epithelial cells. The thymus is generally the first organ to become lymphoid in teleosts (Castillo, 1990; Ellis, 1977; Secombes et al., 1983), although in some species, principally marine fish, the kidney contains hematopoietic tissue before mature thymic lymphocytes are evident (Padrós and Crespo, 1996; Tatner, 1996; Zapata et al., 1996; Zapata et al., 1997). In trout, before hatching, (which occurs at approximately 3 weeks pf ), the kidney contains only developing erythrocytes and myeloid cells 334 WILLETT ET AL. (Castillo, 1990). Preliminary in situ hybridization of kidney tissue shows rag1-positive cells at hatching, when the organ contains cells that are identifiable as lymphoid by EM (Hansen and Zapata, 1998, and unpublished results). In trout, however, both IgM-expressing cells (Castillo et al., 1993) and Ig VH expression are detected much earlier (J. Hansen, personal communication). It is possible that, in trout, a few B cells appear elsewhere in the early embryo before the appearance of lymphocytes in the kidney. Our results confirm the late appearance of lymphoid cells in the zebrafish kidney (Figs. 8, 9; Willett et al., 1997b). Although developing erythrocytes and granulocytes occur in the pronephros from 96 hours onward, the first lymphocytes and lymphoblasts appear later than 2 weeks pf. Consistent with these findings, expression of Ig heavy chain in zebrafish remains at low levels until two weeks pf, when it increases slightly. It then increases dramatically at 3 weeks pf (N. Danilova, personal communication). The nature of the relationship between the lymphoid cell populations of thymus and kidney during teleost ontogeny has been controversial. It has previously been proposed that stem cells migrate from the pronephros to the thymic rudiment to mature and differentiate (Ellis, 1977; Nakanishi, 1991; Padrós and Crespo, 1996). However, these authors did not consider the existence of other sites of hematopoiesis earlier than the pronephros. Re-evaluation of results presented by Padrós and Crespo (1996) suggest the existence of an ICM in the turbot embryo. Our results suggest that lymphoid progenitors found in the thymic primordium of 72 hour-old zebrafish do not come from the pronephros, which initiates its hematopoietic activity between 72 and 96 hours. It is possible that lymphoid progenitors colonizing the thymic primordium derive from the early hematopoietic centers, e.g., the ICM and/or putative AGM region (the ventral part of dorsal aorta). On the basis of indirect and morphological evidence, a thymic contribution to the lymphoid cell populations that appear in the kidney during teleost ontogeny has been suggested (Grace and Manning, 1980; Josefsson and Tatner, 1993; Nakanishi, 1991). However, removal of the thymus in rainbow trout at 4 to 24 days posthatching has no effect on the lymphocyte population in the kidney (Manning et al., 1982). It is likely, therefore, that colonization of the kidney by early hematopoietic progenitors is similar to, but independent from, population of the thymus. Thompson et al. (1998) have proposed, but not shown, that progeny of the c-mybexpressing cells of the dorsal aorta may colonize the kidney. Hematopoiesis in zebrafish is typical of teleosts, and more broadly, of vertebrates in general. The first site of hematopoeisis, the ICM, is derived from lateral mesoderm and provides primitive progenitors, giving rise to embryonic erythrocytes. The posterior ICM appears to maintain hematopoietic potential for up to 4 days pf. The dorsal aorta, a structure physically related to the ICM, but developmentally later, may be the zebrafish equivalent of the AGM, the first site of definitive hematopoiesis in other vertebrates. Cells in this region express transcription factors known to be important for definitive hematopoiesis in mammals. Furthermore, definitive hematopoiesis must begin at a location other than the kidney, since lymphocytes appear in the thymus before establishment of hematopoietic tissue in the kidney. Lineage tracing experiments are required to establish the source of lymphocytes that populate the thymus, an experiment that will be facilitated by the embryological advantages of zebrafish. EXPERIMENTAL PROCEDURES Animals Wild-type zebrafish, Danio rerio, Tübingen strain, were obtained from C. Nüsslein-Volhard (Tübingen, Germany) and were reared at 28°C as described (Culp et al., 1991; Lin et al., 1992). Light and Electron Microscopy Wild-type zebrafish, from 5-somite stage (about 11 hpf) to 3 weeks (11.5, 16, 20, 24, 34, 42, 72, 96 hr, and 1, 2, and 3 weeks post-fertilization), at least 3 fish from each developmental stage, were fixed for 3–5 hours in 2.5% glutaraldehyde/0.1M sodium caccodylate at 4°C, washed for equal time in 0.1M sodium caccodylate/5% sucrose, and post-fixed for 2 hr in dark with 1% osmium tetroxide/0.1M sodium caccodylate. After washing in 0.1M sodium caccodylate/5% sucrose, the fish were dehydrated quickly in increasing concentrations of ethanol to 100%. Fish were then washed twice with propylene oxide, 5 min each. Propylene oxide was replaced with a 2:1 mixture of propylene oxide:PB 812 (PB 812:24%(w/v) Polybed 812 resin, 50% (w/v) dodecenyl succinic anhydride, 1% (v/v) 2,4,6-tris(dimethylamino-methyl) phenol; all from PolySciences, Inc., Warrington, PA) overnight at room temperature, then a 1:1 mixture of the same components for 10 hr, and finally pure PB 812 overnight. Blocks were polymerized at 70°C overnight in fresh PB 812. Sections were obtained with a Reichert OM-U3 ultramicrotome. Semi-thin sections stained with an alkaline solution of 1% toluidine blue were used for light microscopy. Ultra-thin sections, obtained from selected areas on the semi-thin sections, were double stained with uranyl acetate and lead citrate, examined and photographed in a JEOL 10.10 electron microscope. ACKNOWLEDGMENTS We would like to thank Drs. Nadia Danilova, Valerie Hohman, and Lisa Steiner for thoughtful comments. A.C. and A.G. 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