A Fate Map of the Vascular Endothelial and Red Blood Cell Lineages in Xenopus Laevis

Developmental Biology 209, 352–368 (1999) Article ID dbio.1999.9245, available online at http://www.idealibrary.com on Elucidating the Origins of the Vascular System: A Fate Map of the Vascular Endothelial and Red Blood Cell Lineages in Xenopus laevis

Kenna R. Mills, Deborah Kruep, and Margaret S. Saha1 Department of Biology, College of William and Mary, Williamsburg, Virginia 23187

Required to supply nutrients and oxygen to the growing embryo, the vascular system is the first functional organ system to develop during vertebrate embryogenesis. Although there has been substantial progress in identifying the genetic cascade regulating vascular development, the initial stages of vasculogenesis, namely, the origin of vascular endothelial cells within the early embryo, remain unclear. To address this issue we constructed a fate map for specific vascular structures, including the aortic arches, endocardium, dorsal aorta, cardinal veins, and lateral abdominal veins, as well as for the red blood cells at the 16-cell stage and the 32-cell stage of Xenopus laevis. Using genetic markers to identify these cell types, our results suggest that vascular endothelial cells can arise from virtually every blastomere of the 16-cell-stage and the 32-cell-stage embryo, with different blastomeres preferentially, though not exclusively, giving rise to specific vascular structures. Similarly, but more surprisingly, every blastomere in the 16-cell-stage embryo and all but those in the most animal tier of the 32-cell-stage embryo serve as progenitors for red blood cells. Taken together, our results suggest that during normal development, both dorsal and ventral blastomeres contribute significantly to the vascular endothelial and red blood cell lineages. © 1999 Academic Press Key Words: fate map; vasculogenesis; endothelial; blood cell; ventral blood islands; X-msr; globin; lineage.

INTRODUCTION Flamme et al., 1997; Hanahan, 1997; Kumar et al., 1998; Yancopoulos et al., 1998). In order to sustain the process of embryonic growth and However, the early embryology of vascular develop- differentiation, it is essential that the vertebrate embryo ment, that is, the process by which vascular endothelial develop a functional circulatory system during the early cells are ﬁrst generated from the mesoderm, has been less stages of development. This involves not only the forma- intensively studied. While the differentiation of endothe- tion of a beating heart, but also the generation of a lial cells does not depend on gastrulation, at least in coordinated network of blood vessels that supplies oxy- avians (Christ et al., 1991), the identity of the progenitor gen and nutrients to the various organ systems (reviewed cells in the early embryo that eventually give rise to in Risau, 1995; Wilting et al., 1995b; Baldwin, 1996; vasculature remains unclear. Early views, such as those Risau, 1997). Although normally quiescent in the adult of His (1900), suggesting that vasculogenesis was initi- except in the cases of wound healing and reproduction, ated extraembryonically in the yolk sac with the vessels the vascular system demonstrates a high proliferative subsequently invading the embryo proper were soon potential under conditions such as tumorigenesis and contradicted with evidence that the vasculature also diabetes (reviewed in Folkman, 1995; Breier et al., 1997). arose from the splanchnic mesoderm within the embryo Due to its clear importance in both embryonic develop- (Sabin, 1917). Because of the close association of angio- ment and pathogenesis, there has been substantial blasts and red blood cells these early studies hypoth- progress in identifying the key players in the genetic esized the existence of a hemangioblast—a bipotential cascade regulating vascular development (reviewed in Folkman and D’Amore, 1996; Beck and D’Amore, 1997; precursor cell that could give rise to either a blood cell or a vascular cell (Murray, 1932). Several lines of evidence provided by more recent studies lend support to the 1 To whom correspondence should be addressed. hemangioblast hypothesis. These include the existence

0012-1606/99 $30.00 Copyright © 1999 by Academic Press 352 All rights of reproduction in any form reserved. Fate Map of Vascular and Blood Cell Lineages in Xenopus 353 of several monoclonal antibodies that recognize an epitope common to blood cells and endothelial cells (Peault et al., 1983; Pardanaud et al., 1987) and the critical role of a number of genes including Flk-1 (Shalaby et al., 1995, 1997), VEGF (Ferrara et al., 1996; Carmeliet et al., 1996), SCL/Tal-1 (Drake et al, 1997; Liao et al., 1998; Visvader et al., 1998), and cloche (Liao et al., 1997) in both vascular and hematopoietic development. How- ever, other studies employing orthotopic and heterotopic transplants of chick and quail tissues as a means of fate mapping demonstrate that vascular cells, particularly those derived from the paraxial regions of mesoderm, do not share a common origin with hematopoietic cells (Pardanaud et al., 1996). Other experiments using a similar strategy suggest a broadly dispersed origin for vascular endothelial cells that does not correlate with the FIG. 1. Identification of injected blastomeres. Blastomeres were accepted ventral origins for blood cells (Noden, 1990). designated according to Moody (1987a,b). Although a fate map for individual blastomeres would more precisely determine the origin of vascular endothelial cells, embryo size and accessibility at these developmental stages precludes such an analysis in avian and murine MATERIALS AND METHODS systems. Fate maps have been constructed for zebrafish (Kimmel et al., 1990; Lee et al., 1994) and Xenopus (Moody, Collection and Selection of Embryos 1987a,b; Dale and Slack, 1987), but these studies focused on the major tissue systems within the early embryo and did Mature X. laevis frogs were obtained from Xenopus I (Ann Arbor, not specifically examine the vascular endothelial or red Michigan). Fertilized eggs were obtained by injecting pigmented blood cell lineages or they were performed at later blastula frogs subcutaneously with human chorionic gonadotropin (400 u for males and 600 u for females) and placing a male and a female stages. The purpose of the present study is to define the together overnight in an enclosed chamber. Fertilized eggs were contributions of individual blastomeres to the network of collected the following morning and protective jelly coats were vasculature as well as to the red blood cell lineage, infor- removed from the eggs by digestion with 2% L-cysteine and 0.18 M mation that will serve as the basis for examining the tissue NaOH at room temperature for 5 min. Embryos were then rinsed interactions governing vascular development. Toward this three times with 0.10ϫ Normal Amphibian Medium (NAM) (Slack, end we present a fate map for the aortic arches, endocar- 1984) and subsequently placed in petri dishes. Embryos at the 16- or dium, cardinal veins, dorsal aorta, lateral abdominal veins, 32-cell stage (stage 5 and stage 6, respectively; all stages according and red blood cells in Xenopus laevis at the 16- and 32-cell to Nieuwkoop and Faber, 1967) were selected for injection based stages—the latest stages at which cells are still reliably upon the presence of stereotypical pigmentation and regular cleav- identifiable but have already begun to acquire regional age following the criteria set forth by Moody (1987a,b). specification. In order to identify cell types unambiguously, we have chosen to utilize X-msr (Devic et al., 1996; Drys- Lineage Labeling dale et al., 1997; Cleaver et al., 1997), a member of the G-protein-coupled receptor family isolated from X. laevis, Ten minutes before as well as during the injection, embryos were cultured in a solution of 0.75ϫ NAM, 5% w/v Ficoll. For a to specifically identify the vascular endothelial cells and ␮ ␤ given stage, one blastomere of each embryo was injected with 7 l -globin to identify red blood cells in the ventral blood of 45 mg/ml tetramethylrhodamine-linked dextran (RLDx; Mo- islands (Meyerhof et al., 1984). lecular Probes; see Fig. 1 for identity of injected blastomeres). Our data suggest that vascular endothelial cells can arise Blastomeres were initially given alpha-numerical designations (as from virtually every blastomere of the 16-cell stage and the listed in Tables 1 and 2) different from previously published fate 32-cell-stage embryo, with different blastomeres preferen- maps so that data analysis could be conducted without the knowl- tially, though not exclusively, giving rise to specific vascu- edge of the particular blastomere being analyzed. Injections were lar structures. Similarly, but somewhat surprisingly, every performed at an injection pressure of 10 psi for 100 ms using a fine ϫ blastomere in the 16-cell-stage embryo and all but the most needle from a 1.0 0.75-mm capillary tube (Medical Systems animal tier of blastomeres in the 32-cell-stage embryo serve Corp., PLI-C8) pulled (World Precision Instrument, PUL-1) with a “delay setting” of 3 and “heat setting” of 9. Embryos were then as progenitors for red blood cells. Furthermore, our results stepped down through 0.75ϫ NAM (for five cleavages) and 0.375ϫ suggest that while certain vascular endothelial cells can NAM (for 2 h) to 0.10ϫ NAM (at 16°C until fixation). Injected originate from a hemangioblast (a common precursor for embryos were transferred to fresh 0.75ϫ NAM, 5% w/v Ficoll to endothelial and blood cells), there may also be an alterna- inhibit cytoplasmic leakage from the injection site. Any embryos tive pathway for vascular endothelial determination and which exhibited blebbing, leakage, cell death, or irregular cleavage differentiation. following the injection were discarded from the study. The remain-

FIG. 2. Identiﬁcation of vascular structures and ventral blood islands. Whole-mount in situ hybridization was performed on hatching- stage embryos (stage 33/34) using (A) X-msr to identify vascular tissue and (B) ␤-globin to identify the ventral blood islands. (C) Schematic diagram indicating the location of structures examined in this study. aa, aortic arches; acv, anterior cardinal vein; da, dorsal aorta; endo, endocardium; lav, lateral abdominal vein; pcv; posterior cardinal vein; sa, somitic arteries; tb, tailbud; vbi, ventral blood islands.

ing viable embryos were then fixed at stages 33–34 (early hatching embryos were placed for 15 min at room temperature in 100% stages) in 1ϫ MEMFA (Harland, 1991) for 1.5 h and subsequently EtOH:xylene (1:1), for 15 min at 60°C in 100% xylene, for 30 min stored in 100% ethanol at Ϫ20°C. Sibling embryos, that is, unin- at 60°C in xylene:paraplast (1:1), and finally for2hin100% jected controls, as well as embryos injected with water which were paraplast at 60°C prior to embedding. Ten-micrometer transverse used as controls for the injection procedure, were treated in a sections were placed on microscope slides prepared by treatment manner identical to the injected embryos. with Mayer’s albumin fixative (Humason, 1979), cleared with xylene, and coverslipped in 100% Permount (Fisher) for bright-field and fluorescence microscopy. Probe Synthesis, In Situ Hybridization, and Histology Fate Mapping In order to identify vascular and hematopoietic cells unambiguously, molecular markers were employed (Fig. 2). X-msr (Devic et Histology was analyzed using the Olympus BX60 System micro- al., 1996; independently cloned in our lab as Xangio, GenBank scope and the BX-FLA reflected-light fluorescence attachment. Accession No. U72029) was used as a marker for vascular tissue RLDx was visualized using a U-MWG cube with an excitation and ␤-globin was employed as a marker for red blood cells (Meyer- range of 510–550 nm. All specimens were first scanned at 40ϫ or hof et al., 1984). Plasmid DNA for X-msr and ␤-globin was isolated 100ϫ; those embryos showing strong RLDx fluorescence and a using a standard alkaline lysis and PEG precipitation method robust in situ hybridization signal were examined for tissue dem- described by Sambrook et al. (1989). Plasmid DNA for X-msr and onstrating colocalization of RLDx with the in situ hybridization ␤-globin was linearized with BamHI and transcribed with T7 RNA signal for ␤-globin or X-msr. Magnification was then increased to polymerase to generate antisense probes for in situ hybridization. 400ϫ, at which a considerable degree of cellular resolution is Whole-mount in situ hybridization was carried out essentially as possible, to confirm colocalization of the in situ hybridization described by Harland (1991). For histological analysis, embryos signal and RLDx on a cellular basis. This was accomplished using were first dehydrated in sequential washes of 50, 70, and 100% (a) the “wide green” cube in conjunction with low bright-field light EtOH for 10 min each. After repeating the last 100% wash, to visualize RLDx and the in situ hybridization signal simulta-

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. Fate Map of Vascular and Blood Cell Lineages in Xenopus 355 neously and (b) the “combine images” feature of PhotoShop. For the results for structures not specifically analyzed by X-msr five specific structures were analyzed: endocardium, aortic Moody or Dale and Slack, namely, the vasculature and red arches, cardinal veins, dorsal aorta, and the lateral abdominal veins. blood cells. The results for aortic arches, endocardium, At least 10 embryos were analyzed for each blastomere for both the cardinal veins, dorsal aorta, lateral abdominal veins, and red vascular endothelial and the red blood cell lineages for the 16-cell- blood cells are presented below as well as in Tables 1 and 2. stage embryo, and at least 5 embryos were analyzed for each blastomere of the 32-cell-stage embryo. We also estimated the extent to which a given blastomere contributed to a particular Aortic Arches structure (e.g., endocardium or ventral blood islands). This was accomplished by using an ocular grid to measure the area of the in Ventrolateral to the anterior notochord, five pairs of situ hybridization signal, to determine the area of a given structure, aortic arches branch from the ventral aorta and unite to and the colocalization of fluorescence with the in situ hybridiza- form the dorsal aorta just dorsal to the pharynx. While each tion signal, to assess the approximate percentage of tissue derived of the blastomeres of the 16-cell-stage embryo contributed from a specific blastomere for that section. These measurements progeny to the aortic arches, blastomeres V1.2, D1.2, D2.2, were made for each relevant section and an average was calculated and D2.1 consistently gave rise to aortic arch tissue with to approximate the percentage of a specific vascular structure that high frequency; 60% of V1.2, 70% of D1.2, 100% of D2.2, was derived from a particular blastomere. These results are pre- and 55% of D2.1 injected blastomeres produced progeny sented in Tables 1 and 2. Because it was not possible to determine the precise volume of tissue contributed from a given blastomere to that populated the aortic arches with V1.2, D1.2, and D2.2 a given structure (because it was extrapolated from the area of blastomeres consistently contributing between 36 and 65% tissue covered on the slides), we stress that these measurements are of the aortic arch tissue and D2.1 blastomeres providing semiquantitative. between 10 and 35% (Table 1). There is a clear anterior– Other structures (brain, notochord, pharynx, myocardium, posterior polarity with respect to origins of the aortic retina, lens, epidermis, lateral plate mesoderm, somites, and hind- arches: D2.1 produced progeny that gave rise, almost exclu- gut) were examined in all embryos and used for purposes of sively, to the most anterior aortic arches while D2.2 dem- comparison with previously constructed maps (Moody, 1987a,b; onstrated the opposite trend and contributed progeny only Dale and Slack, 1987). Photographs were taken using a Spot digital to the posterior aortic arches, that is, those closest to the camera (Diagnostic Instruments, Inc.). Fluorescent images were endocardium. The remainder of the blastomeres had deriva- overlaid on bright-field images using the combine images function in the Spot camera program (version 1.1.02). tives throughout the aortic arches. At the 32-cell stage the aortic arch progenitors remained widely distributed with all blastomeres except D1.1.1, V1.1.1, V2.1.1, and V2.2.1 con- RESULTS tributing to this tissue. Expectedly, it was the daughter cells of those blastomeres in the 16-cell-stage embryo that con- Nonvascular Tissues sistently contributed progeny to aortic arch tissue that For purposes of comparison with other published fate likewise remained primary contributors of progeny to the maps we have examined the contributions of each of the aortic arches at the 32-cell stage (Table 2). blastomeres of the 16-cell- and 32-cell-stage embryos to selected nonvascular structures, including brain, noto- Cardinal Veins chord, pharynx, myocardium (Tables 1 and 2), cement gland, retina, somites, spinal cord, brain, epidermis, and The cardinal veins are generally divided into the anterior hindgut (data not shown). The fate maps described in the and the posterior cardinal veins. The anterior cardinal vein present study are consistent with the data presented by extends ventrolaterally along the brain floor and runs hori- Moody (1987a,b) and Dale and Slack (1987) for all structures zontally along the base of the brain before entering the analyzed in both studies. Comparing this study to that common cardinal veins at the level of the heart. The presented by Moody, which like this study analyzed data as posterior cardinal vein also runs horizontally, ventrolater- the frequency with which a given blastomere contributed to ally to the notochord from the tailbud to the common specific structures, the two agree for most structures to cardinal veins. All of the blastomeres of the 16-cell-stage within approximately 25%, a significant degree of consis- embryo served as progenitors to cardinal veins with the tency given the natural variation in cleavage patterns. vegetal blastomeres V2.1, V2.2, and D2.2 serving as primary Several apparent discrepancies reside not in the primary contributors; 67% of V2.1, 90% of V2.2, and 90% of D2.2 data (that is, the tables) but rather in the presentation of the injected blastomeres contributed to this tissue and each of data. For example, for heart we have distinguished between these blastomeres consistently provided over 35% of cardi- endocardium and myocardium, but for neural tissue we nal vein tissue (Table 1; Figs. 3A–3C and 3G–3I). As with have not distinguished among the various anterior– the aortic arches, there is a clear anterior–posterior polarity posterior divisions of the brain as does Moody (1987a,b). In with respect to the origin of the cardinal veins, with the short, the data presented in Tables 1 and 2 (and structures progeny of D2.1 and D2.2 contributing almost exclusively for which data are not shown) are generally equivalent to to the anterior cardinal vein and V2.1 to the posterior that presented by Moody (1987a,b) and Slack (1987). This cardinal vein. Consistent with the fate-mapping results of consistency provided some assurance of accuracy regarding the 16-cell-stage embryo, all blastomeres of the 32-cell-

TABLE 1a Progeny of Dorsal Blastomeres (16-Cell Stage)

Blast. Emb. BR NC PH MY EN CV AA LAV DA Emb. VBI

D1.1 A11 ϩϩϪϩϩϩϩϩ Ϫ ϩ ϪA1E1 ϩ A13 ϩϩϩ * ϩϩϩ ϩ ϩ Ϫ ϩϩ Ϫ Ϫ A1E4 Ϫ A15 ϩϩϩϩϩϩϪϪϪ Ϫ Ϫϩϩ,a A1E5 ϩ A16 ϩϩϩ ϩϩϩ ϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩ A1E6 ϩ A17 ϩϩϩ ϩϩϩ ϩϩ Ϫ ϩ Ϫ Ϫ Ϫ ϩϩ,a A1E7 * A18 ϩϩϩ ϩϩϩ ϩ ϩϩ ϩϩ Ϫ Ϫ Ϫ ϩϩ A1E8 Ϫ A19 ϩϩϩ ϩϩϩ ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ,a A1E9 * A110 ϩϩϩ ϩϩϩ ϩϩϩ Ϫ Ϫ Ϫ ϩ ϩ ϩ,a A1E1 * A111 ϩϩϩ ϩϩϩ ϩϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ,a A1E1 Ϫ A112 ϩ ϩ ϩϩ ϩϩ ϩϩ Ϫ ϩ Ϫ Ϫ A1E1 * A113 ϩϩϩ ϩϩϩ ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩ,a A1E1 Ϫ D1.2 A22 ϩϩϪϩϪϪϪ* ϪϪA2E1 Ϫ A23 ϩϩϩϪϩϪϪϪϪϪA2E2 Ϫ A24 ϩϪϪϪϪ* ϩϩϩ Ϫ Ϫ A2E5 ϩ A25 ϩϩϩϪϩϪϩϩ,a ϩϩϪϩ,a A2E6 Ϫ A26 ϩϩ ϩϩϩ ϩϩ Ϫ ϩϩ Ϫ Ϫ Ϫ Ϫ A2E8 Ϫ A27 ϩϩϩϩϩϩϪϪϩϩϪϩ,a A2E9 ϩ A28 ϩϪϪϪϪϪϩϩϪϪA2E1 ϩ A29 ϩϩϩϩϪϪϪϪϪϪϪA2E1 ϩ A210 ϩϩ ϩϩ Ϫ Ϫ Ϫ Ϫ ϩ Ϫ Ϫ A2E1 ϩ A211 ϩϩϩ ϩ ϩ ϩ * Ϫ ϩϩ Ϫ ϩϩϩ A2E1 * D2.1 V11 Ϫ ϩ ϩϩ ϩϩ ϩϩ Ϫ Ϫ Ϫ ϩ V1E1 ϩ V12 ϪϪϩϩϩϪϩϪϪϪϪV1E2 Ϫ V13 Ϫ Ϫ ϩ ϩϩϩ Ϫ ϩϩ,a ϪϩϩϩV1E4 ϩϩ V14 Ϫ Ϫ ϩϩϩ ϩϩϩ Ϫ Ϫ ϩ,a ϩϩ Ϫ V1E5 * V15 Ϫϩϩϩ**ϪϪϪϪV1E6 ϩϩ V16 Ϫ Ϫ ϩϩϩ ϩϩϩ Ϫ Ϫ ϩ,a ϩϩ Ϫ V1E7 ϩϩ V17 ϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩ Ϫ ϩ,a ϩϩ Ϫ V1E8 ϩ V18 Ϫ Ϫ ϩϩϩ ϩϩϩ ϩ Ϫ ϩ,a ϩϩV1E9 ϩ V111 ϩϩϩϩϩϪϪ Ϫ Ϫ ϪV1E1 ϩϩ V112 Ϫ Ϫ ϩϩ ϩϩ Ϫ Ϫ ϩ,a ϩϩ Ϫ V1E1 ϩ V113 ϪϪϩϩ**ϩ,a ϩϩϩϪV1E1 Ϫ D2.2 V21 ϩϩϪϩϩϩϩϩ,a ϩϩ,p ϪϪV2E1 ϩϩϩ V22 * ϪϪϩϩ* ϩϩ,a ϩϩ,p * Ϫ V2E2 Ϫ V23 Ϫ ϩϩ Ϫ ϩϩϩ ϩϩ Ϫ ϩϩϩ ϩϩ ϩ V2E3 ϩ V25 ϪϩϩϪϩϩϪϩϩ,a ϩϩ,p Ϫ * V2E4 Ϫ V27 Ϫ Ϫ ϩ ϩϩϩ Ϫ ϩϩ,a ϩ * ϩ V2E7 ϩ V29 ϩϪ * ϩϩ * ϩϩ,a ϩϩϩ ϩϩϩ Ϫ V2E8 ϩϩ V215 ϩϪϪϩϪϩ,a ϩ,p ϩϩ ϩ V2E9 ϩϩ V216 ϪϪϪϩϩϩϩ,a ϩϩ,p ϩϩ ϩ V2E1 Ϫ V217 ϪϪϩϩϩ* ϩϩ,a ϩϩϩ ϩϩϩ * V2E1 Ϫ V218 ϩ ϩ ϩϩ ϩϩ ϩϩ ϩϩ,a ϩϩ,p ϩϩ ϩ V2E1 Ϫ

stage embryo contributed to the cardinal veins except diverticulum. It is composed of a closed ring of endocardium D2.1.2. Progeny of the ventral vegetal blastomeres as well (vascular endothelial cells distinguishable by X-msr expres- as of the dorsal vegetal blastomere D2.2.1 were the primary sion) and a surrounding myocardium. At the 16-cell-stage contributors to cardinal vein tissue (Table 2). endocardial cells consistently derive from three blastomeres: 45% of D1.1, 55% of D2.1, and 70% of D2.2 injected blastomeres gave rise to progeny contributing to the endocardium Endocardium with minor contributions from V1.1 and D1.2 (Figs. 3D–3F). Surrounded by the myocardium, which gives rise to the The majority of contributing blastomeres provided no more contractile tissue of the heart, the endocardium forms the than 35% of endocardial tissue (Table 1). Unlike the other inner endothelial lining of the heart. At hatching stages the vascular structures examined in this study, the endocardium heart lies at the base of the foregut, anterior to the liver appears to be the only one in which the entire circumference

TABLE 1b Progeny of Ventral Blastomeres (16-Cell Stage)

Blast. Emb. BR NC PH MY EN CV AA LAV DA Emb. VBI

V1.1 A4 2 Ϫ Ϫ Ϫ Ϫ ϩϩϩ ϩϩ Ϫ ϩϩ Ϫ A4E1 ϩ A4 5 ϩϪϪϪ Ϫ * ϩϪ ϪA4E2 ϩϩ A4 7 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪA4E4 ϩ A4 8 ϩϪϪϪ Ϫ * ϪϪ ϪA4E5 ϩ A4 9 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪA4E6 ϩϩ A414 ϩϪϩϪϪ ϩϩϩϪϪA4E7 Ϫ A417 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪA4E9 ϩ A421 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪA4E1 ϩϩ A422 ϪϪϪϪ Ϫ ϩ Ϫ Ϫ ϪA4E1 ϩ A425 ϪϪϪϪ Ϫ Ϫ ϩ Ϫ ϪA4E1 Ϫ V1.2 A33 ϩϪϪϪ Ϫ ϩ ϩϩϪ ϪA3E1 ϩ A34 ϪϪϪϪ Ϫ ϩ ϩ Ϫ ϪA3E2 ϩϩ A36 ϩϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪA3E3 Ϫ A38 ϪϪϩϪ Ϫ ϩ ϩϩϪ ϪA3E4 ϩ A39 ϩϪϩϪ Ϫ Ϫ Ϫ Ϫ ϪA3E5 * A310 ϩϪϪϪ Ϫ Ϫ ϩϩϪ ϪA3E6 * A311 ϩϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪA3E7 Ϫ A314 * Ϫ * ϪϪ ϩ,a ϩϩ Ϫ Ϫ A3E8 Ϫ A316 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪA3E9 Ϫ A319 ϪϪϪϪ Ϫ ϩ,a ϩϪ ϪA3E1 Ϫ V2.1 V4 1 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪV4E1 Ϫ V4 2 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϩ,p V4E2 ϩϩ V4 3 ϪϪϪϪ Ϫ Ϫ Ϫ Ϫ ϪV4E3 ϩ V4 4 ϪϪϪϪ Ϫ ϩϩ,p ϪϪϩϩ,p V4E4 ϩ V4 6 Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ,p ϩϩ Ϫ ϩ V4E5 ϩϩ V4 8 Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ,p ϩϩ ϩϩ,p Ϫ V4E6 ϩ V410 ϪϪ* ϪϪ Ϫ ϪϪϩϩ,p V4E7 ϩ V412 Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ,p ϩϩϩϩV4E8 ϩ V413 Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ,p Ϫ * ϩ V4E9 ϩϩ V415 ϪϪϪϪ Ϫ ϩϩ,p Ϫϩ ϩV4E1 Ϫ V416 Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩϩ,p Ϫϩ,p Ϫ V4E1 ϩ V417 ϩ Ϫ Ϫ Ϫ Ϫ ϩϩϩ,p Ϫϩ ϪV4E1 * V2.2 V32 ϩϩ Ϫ Ϫ ϩϩ Ϫ ϩϩ,a ϩϩ ϩ ϩ V3E1 Ϫ V34 ϩϪϪϪ Ϫ ϩϩϩϩϩ ϩV3E2 ϩϩ V35 ϪϪϪϪϪ ϩϩϪϪ * V3E3 ϩϩ V36 ϪϪϪϪϪ ϩϩϪϩ * V3E4 ϩ V37 ϪϪϪϪϪ ϩϩϪϩϪV3E5 ϩ V38 ϪϪϪϪϪ ϩϩϪϪϪV3E6 * V39 ϪϪϪϪ Ϫ Ϫ ϩ ϩ ϪV3E8 Ϫ V310 ϪϪϪϪϪ ϩϩϪϪϩV3E9 ϩ V311 ϪϪϪϪϪ ϩϩϪϩϪV3E1 ϩϩ V315 ϪϩϪϪϪ ϩϩϪϩϩV3E1 ϩ

Note. Tables correspond to data collected for all of the blastomeres of the 16-cell- and 32-cell-stage embryos. A minimum of 10 embryos were analyzed for the 16-cell stage and 5 embryos for the 32-cell stage. Separate embryos were analyzed for X-msr and ␤-globin expression; the embryos listed in column 2 and column 12 were from different pools. An approximate determination of the percentage of tissue in a speciﬁc structure contributed by a given blastomere is designated as follows: *less than 10%; ϩ, 10–35%; ϩϩ, 36–65%; ϩϩϩ, 66–100%. A lowercase “a” indicates that labeled tissue was found almost exclusively in the anterior region of a speciﬁc organ; a lowercase “p” indicates that labeled tissue was located almost exclusively in the posterior region of the structure. Structures are designated as follows: BR, brain; NC, notochord; PH, pharynx; MY, myocardium; EN, endocardium; CV, cardinal veins; AA, aortic arches; LAV, lateral abdominal veins; DA, dorsal aorta; VBI, ventral blood islands.

of the organ is almost never derived from a single blastomere the endocardium derived from a single blastomere (V1.1, A4 but rather is a mosaic of cells derived from several blas- 2). At the 32-cell stage the dorsal blastomeres continue to tomeres. In all embryos examined there was only a single case serve as the primary progenitors of endocardial tissue with a in which the entire circumference in a given section (10␮m) of minor contribution from V1.1.1 (Table 2).

TABLE 2a Progeny of Dorsal Blastomeres (32-Cell Stage)

Blast. Emb. BR NC PH MY EN CV AA LAV DA Emb. VBI

D1.1.1 B2e1 ϩϩϩϩϪϪϪϪϩϪB2E1 Ϫ B2e2 ϩϩ Ϫ Ϫ * ϪϪ Ϫ Ϫϩ,a B2E2 Ϫ B2e3 ϩϩϪ **ϩ,a ϪϪϪB2E3 Ϫ B2e4 Ϫ * ϩ Ϫ ϩϩ ϩϩ,a Ϫϩϩ*,a B2E4 Ϫ B2e5 ϩϩ ϩ ϩ Ϫ Ϫ Ϫ Ϫ Ϫ ϩϩ,a B2E5 Ϫ B2e6 ϩϩ ϩϩ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ ϩ B2E6 Ϫ B2e7 ϩϩ * ϪϪϪϪϪϩB2E7 Ϫ B2E8 Ϫ B2E9 Ϫ B2E1 Ϫ D1.1.2 A2e1 ϩϩ ϩ Ϫ * Ϫϩϩ,a ϩϩ ϩ ϩϩ A2E1 * A2e2 ϩϩ ϩϩ ϩ Ϫ ϩ Ϫ ϩϩ Ϫ Ϫ A2E2 ϩ A2e3 ϩϩ Ϫ ϩ ϩϩ ϩ Ϫ Ϫ Ϫ ϩϩ A2E3 ϩ A2e4 ϩϩϩϩϩϪϩϩϪϪϩϩA2E4 Ϫ A2e5 Ϫ **ϩϪϪϪϩϪA2E5 Ϫ A2e6 ϩϩϪϪϪϩϪϩϩϪϪA2E6 Ϫ A2e7 ϩϩ * ϩϩϩϪϪϩϩϪϩϩA2E7 Ϫ A2e9 ϪϩϪϪϪϩϩϪϪϪA2E8 * A2e8 ϩϩ ϩ **ϩ *,a ϩϩ Ϫ Ϫ D1.2.1 B1e1 ϩϪϪϪϪϩϩ,p ϩϪϩϩB1E1 Ϫ B1e2 ϩϩ ϩϩ ϩϩ ϩϩ ϩϩ Ϫ ϩϩ Ϫ ϩ B1E2 Ϫ B1e3 ϪϪϪϪϪϩϩϪϩϩϪB1E3 Ϫ B1e4 ϩϩϩϪϪϩ,a ϪϪϪB1E4 Ϫ B1e5 ϩϩϪϪϪϩϩϩϩϪϪB1E5 * B1e6 ϩϩ ϩϩ Ϫ Ϫ Ϫ *,a ϪϪϪB1E6 * B1e7 ϩϩϪϪϪϪϩϩ,a ϩϩ Ϫ Ϫ B1E7 Ϫ B1e8 * * ϪϩϩϪϩϩ,a ϩϩ Ϫ Ϫ B1E8 Ϫ B1e9 ϩϩϪϪϪϪϪϩϩϪ B1e10 ϩϩϪϩϪϪϩϩ,a ϩϩ Ϫ Ϫ D1.2.2 A4e1 ϩϪϪϪϪϪϩϪϪA1E1 ϩ A4e2 ϩϪϩϪϪϪϩϩϪϪA1E2 Ϫ A4e4 ϩϩϩϪϪϩϩ,p ϪϪϪA1E3 ϩ A4e5 ϪϪϪϪϪϪϪϪϪA1E4 ϩ A4e6 ϩϪϪϪϪϪϪϪϪA4E1 Ϫ A4e7 Ϫ * ϩϪϪϪϪϪϪA4E2 Ϫ A4e8 ϩϪϪϪϪϪϪϪϪA4E3 Ϫ A4e9 ϪϪ * ϪϪϪϪϪϪA4E4 Ϫ A4e10 * ϩϪϪϪϪϪϪϪA4E5 Ϫ A4e11 ϩϪϩϪϪϪϪϪϪA4E6 Ϫ A4e12 ϩϪϩϪϪϩϩ,p ϪϪϪ D2.1.1 G2e1 Ϫ ϩ Ϫ ϩϩ ϩϩ Ϫ ϩ Ϫ Ϫ G2E1 Ϫ G2e2 ϪϪϪϪϪϪϪϩϪG2E2 ϩ G2e3 Ϫ ϪϩϩϪϪ*,a ϪϩϪG2E3 Ϫ G2e4 ϩϪϪϩϪϪϪϪϪG2E4 ϩϩ G2e5 ϪϪϩϪϪϪϪϩϪG2E5 ϩ G2e6 * ϩϪϪϪϩϩ,p ϩϩ,p ϩϩ Ϫ G2E5 ϩϩϩ G2e7 ϪϪϪϩϩϩϩ,p ϩϩ ϩϩ Ϫ G2E7 ϩ G2e8 ϪϪϩϪϪϪϪϪϪG2E8 ϩϩ G2e9 ϪϪϪϪϪϪϪϩϩϩG2E9 ϩ G2e10 Ϫ * Ϫ ϪϪϪ ϪϩϩϪG2E10 ϩϩϩ G2e11 ϪϪϪ * ϪϪ ϪϩϩϪG2E11 ϩ G2e12 ϪϪ * ϩϩϪϩϩϩϩϪ D2.1.2 H3e1 ϪϩϩϩϩϩϩϪϩϩ * ϩ H2E1 ϩ H3e2 ϪϪϩϩϩ* ϪϩϩϪ * H2E2 ϩϩ H3e3 * ϩϩϩϪϪϪϩϩϩϪH2E3 ϩ H3e4 Ϫ Ϫ ϩϩϩ ϩ ϩ Ϫ ϩ ϩ * H2E4 ϩϩ H3e5 ϩ ϩϩϩϩϩϩϩϪ Ϫ Ϫ ϪH2E5 * H3e6 ϪϪϪϪϪϪϪϪϪH3E1 ϩ

TABLE 2a—Continued

Blast. Emb. BR NC PH MY EN CV AA LAV DA Emb. VBI

H2e1 ϪϩϩϩϪϪϪϪϪϪH3E2 * H2e2 * ϪϩϪϪϪ* Ϫ * H3E3 ϩ H2e3 Ϫϩϩϩ* ϩϩϪϪϪϪ H2e4 ϩϪϩϩϪϪϪϪϪϪ H2e5 ϪϩϩϪϪϪϪϪϪ D2.2.1 G1e1 ϪϪϪϪϪϩϩ,a ϩϩϩϪG4E1 Ϫ G1e2 Ϫ ϩϩϩϩϩϩϩϪ ϩϩϩϩϪG4E2 ϩ G1e3 Ϫ **ϩϩ ϩϩ ϩϩ,a ϩϩϩϩG4E3 Ϫ G1e4 ϪϪ * ϩϩϩϩϩϩ Ϫ ϪG4E4 * G1e5 ϩϩ ϩϩ ϩϩ Ϫ * ϩϩ,a ϩϩ Ϫ Ϫ G4E5 ϩ G4e1 ϩ ϪϩϩϩϪϩ,a ϩϪϪG1E1 Ϫ G4e2 ϩ Ϫ ϩϩϩ ϩϩ ϩ ϩ ϩϩ Ϫ ϩ G1E2 ϩ G4e3 ϩ ϪϩϩϪϪϩϩϩϪϪG1E3 Ϫ G4e4 ϩϪϪϩϪϩ,a ϩϩ ϩϩ Ϫ G1E4 ϩϩ G4e5 ϪϪϪ * Ϫϩ,a ϩϩϩϪG1E5 ϩ D2.2.2 H1e1 ϪϪ * ϪϪϩ * ϪϩH1E1 Ϫ H1e2 Ϫϩϩϩϩϩϩϩ,a * Ϫ * H1E2 Ϫ H1e3 ϩ ϩϩϩ ϩϩϩ ϩϩϩ ϩϩ Ϫ ϩϩϩ ϩ Ϫ H1E3 Ϫ H1e4 ϩϪϪϪϪ*,p ϪϩϪH1E4 Ϫ H1e5 ϪϪϪϪϪϪϪϪϪH4E1 Ϫ H1e6 * * ϩϩϪϪϪϪϪϪH4E2 ϩ H4e1 ϩ ϩϩ ϩϩϩ ϩϩϩ ϩϩ Ϫ ϩ ϩ * H4E3 ϩ H4e2 ϩϪϩϩϪϩ,a ϩϩϩH4E4 Ϫ H4e3 ϩϪϩϪϪϩ,a ϩϩ* H4e4 ϩϩϩ ϩ ϩϩ ϩϩ Ϫ Ϫ ϩ ϩϩϩ Ϫ H4e5 Ϫ * ϩϪϪϪϪϪϪ

Note. See footnote to Table 1.

Lateral Abdominal Veins dorsal aorta, consistently providing no more than 35% of dorsal aorta tissue; only the ventral animal pole blas- The lateral abdominal veins run approximately parallel to tomeres do not contribute to this tissue (Table 1; Figs. the dorsal aorta, but more ventrally traversing the gut 4D–4F). Similar to the cardinal veins, there appears to be region. Lying just underneath the surface of the pigmented a strong anterior–posterior map to this organ with the epidermis, the lateral abdominal veins can be distinguished dorsalmost animal pole blastomeres (D1.1 and D1.2) by the bilateral presence of X-msr signal along the anterior– contributing primarily to the anterior portion of the posterior axis, beginning at the common cardinal vein. The organ and the ventralmost vegetal pole blastomere (V2.1) cells in the lateral abdominal veins were derivatives from contributing to the posterior part of the organ. The the vegetal pole blastomeres; 64% of D2.1, 80% of D2.2, contribution from D2.1, D2.2, and V2.2 corresponds to 50% of V2.1, and 70% of V2.2 injected blastomeres contrib- the area of the structure at the center of the embryo uted to lateral abdominal vein tissue (Figs. 4A–4C). To- approximately between the area of the liver diverticulum gether, D2.1 and D2.2 are responsible for approximately and the end of the hindgut. The lineage at the 32-cell half of the tissue in the lateral abdominal veins (Table 1). At stage is consistent with that of the 16-cell stage with all the 32-cell stage, the vegetal blastomeres remain the most blastomeres except the animal tier blastomeres (V1.1.1, frequent contributors of progeny to the lateral abdominal V1.1.2, V1.2.1, and V1.2.2) (Fig. 5) consistently contrib- veins (Table 2). uting to the dorsal aorta tissue (Table 2).

Dorsal Aorta Ventral Blood Islands The dorsal aorta runs along the anterior–posterior axis The precursors of the hematopoietic system, or the red from the tailbud to the heart directly ventral to the blood cells, are localized to the ventral portion of the notochord and hypochord. At tailbud stages this struc- anterior gut region in tailbud-stage embryos and can be ture has yet to fuse and appears as two distinct tubules at identiﬁed precisely via the expression of globin. Anteri- the roof of the gut. At the 16-cell stage, blastomeres D1.1, orly, the expression pattern begins in a V shape and then D1.2, D2.1, D2.2, V2.1, and V2.2 all contribute to the becomes a solid band as the signal moves posteriorly.

TABLE 2b Progeny of Ventral Blastomeres (32-Cell Stage)

Blast. Emb. BR NC PH MY EN CV AA LAV DA Emb. VBI

V1.1.1 C3e1 ϪϪϪϪϪ Ϫ Ϫ ϩ ϪC3E1 Ϫ C3e2 ϩ ϪϩϩϪϩϩϪ Ϫ Ϫ ϪC3E2 * C3e3 ϪϪϪϪϪ Ϫ Ϫ Ϫ ϪC3E3 Ϫ C3e4 ϪϪϪϪϪϩ,p ϪϪϪC3E4 * C3e5 * ϪϪϪϪ Ϫ Ϫ Ϫ ϪCEE5 Ϫ C3e6 ϪϪϪϪϪϩ,p ϪϪϪC3E6 Ϫ C3e7 * ϪϪϪϪ Ϫ Ϫ Ϫ ϪC3E7 Ϫ C3e8 ϪϪϪϪϪ Ϫ Ϫ Ϫ Ϫ C3e9 ϪϪϪϪϪϩ,p ϪϪϪ C3e10 ϪϪϪϪϪ Ϫ Ϫ Ϫ Ϫ C3e11 ϪϪϪϪϪϩ,p ϪϪϪ V1.1.2 D2e1 ϩϪϪϪϪ ϩ ϩ Ϫ ϪD2E1 ϩ D2e2 ϩϪϪϪϪ ϩ Ϫ Ϫ ϪD2E2 Ϫ D2e3 ϩϪϪϪϪ Ϫ Ϫ Ϫ ϩD2E3 ϩϩ D2e4 ϪϪϪϪϪ Ϫ Ϫ Ϫ ϪD2E4 * D2e5 ϪϪϪϪϪ Ϫ Ϫ Ϫ ϪD2E5 Ϫ D2e6 ϩϪϪ * Ϫ * ϪϪϪD236 ϩ D2e7 ϪϪϪϪϪ Ϫ Ϫ Ϫ ϪD2E7 Ϫ D2e8 ϩϪϪϩϪϩϩ,a ϩϩϩϪD2E8 ϩ D3e1 ϪϪϪϪϪ Ϫ Ϫ Ϫ Ϫ D3e3 ϪϪϪϪϪ *,p ϪϪϪ D3e4 ϪϪϪϪϪ * ϪϪϪ D3e5 ϩϩϪϪϪϪϩ,a ϩϪϪ V1.2.1 C1e1 ϪϪϪ * Ϫ Ϫ ϩϪϪC1E3 Ϫ C1e2 ϩ * Ϫ * Ϫ Ϫ ϪϪϪC1E2 Ϫ C1e3 ϪϪϪϪϪ ϪϩϩϪϪC1E7 Ϫ C1e4 ϪϩϪ * Ϫ Ϫ ϪϪϪC1E4 Ϫ C1e5 ϩϪϪϪϪ Ϫ Ϫ Ϫ ϪC1E5 Ϫ C1e6 ϪϪϪϪϪϩ,a * ϪϪC1E6 Ϫ C1e7 * ϪϪ * Ϫϩ,a ϪϪϪC1E8 Ϫ C1E9 Ϫ C1E1 Ϫ C1E1 Ϫ C1E1 Ϫ C1E1 Ϫ V1.2.2 D1e2 ϪϪϪϪϪ Ϫ Ϫ Ϫ ϪD1E1 ϩ D1e3 ϩϪϪϪϪ ϩ ϩ Ϫ ϪD1E2 Ϫ D1e4 ϪϩϪϪϪϩ,p ϪϪϪD1E3 ϩϩ D1e5 ϪϪϪϪϪ Ϫ Ϫ Ϫ ϪD1E4 * D1e6 ϪϪϪϪϪ Ϫ ϩ Ϫ ϪD1E5 Ϫ D1e7 Ϫ * ϪϪϪϩ,p ϪϪϪD1E6 Ϫ D1e8 ϩϪϪϪϪ ϩϩϩϪϪD1E7 ϩ D1e9 ϪϪϪϪϪϩϩϩϩϪϪ D1e10 ϩϪϪϪϪ ϪϩϩϪϪ D4e11 ϪϩϪϪϪ Ϫ Ϫ Ϫ Ϫ D4e13 ϪϪϪϪϪϩϩ,p ϪϪϪ V2.1.1 F2e1 ϪϪϪϪϪϩϩ,p ϪϩϩϩF2E1 ϩϩ F2e2 ϪϪϪϪϪϩϩ,p Ϫ * ϩ F2E2 ϩϩ F2e3 ϪϪϪϪϪϩϩ,p ϪϩϩϩF2E3 ϩϩ F2e4 ϪϪϪϪϪϩϩ,p ϪϩϩϩF2E4 ϩϩ F3e1 ϪϪϪϪϪϩ,p ϪϪϪF2E5 Ϫ F3e2 ϪϪϪϪϪϩϩ,p ϪϪϪF2E6 ϩϩ F2E7 ϩϩϩ F2E8 ϩϩ F2E9 ϩϩ F2E10 ϩ V2.1.2 E2e1 ϪϪϪϪϪϩϩ,p ϪϪϪE231 Ϫ E2e2 ϪϪϩϪϪϩ,p ϩϪϩE2E2 Ϫ E2e3 ϪϪϪϪϪϩ,p ϪϪϪE2E3 Ϫ

TABLE 2b—Continued

Blast. Emb. BR NC PH MY EN CV AA LAV DA Emb. VBI

E2e4 ϪϪϪϪϪϩϩ,p ϪϪϪE2E4 Ϫ E2e5 ϪϪϪϪϪϩϩ,p ϪϪϩE2E5 * E2e6 ϪϪϪϪϪϩϩ,p ϪϪϪE2E6 Ϫ E2e7 ϪϪϪϪϪϩϩ,p ϪϪϩE3E1 * E2e8 ϪϪϪϪϪϩϩ,p ϪϪϪE3E2 Ϫ E3E3 Ϫ E3E4 ϩϩ E3E5 ϩ V2.2.1 F1e1 ϪϪϪϪϪϩϩ,p ϪϩϩϩF1E1 ϩ F1e2 ϪϪϪϪϪ Ϫ Ϫ Ϫ ϪF1E2 ϩ F1e3 ϪϪϪϪϪϩ,p ϪϩϪF1E3 Ϫ F1e4 ϪϪϪϪϪϩϩ,p ϪϩϩϩF1E4 * F1e5 ϪϪϪϪϪϩϩ,p ϪϪϩF1E5 Ϫ F1e6 ϪϪϪϪϪϩ,p ϪϩϪF1E6 ϩ F1e7 ϪϪϪϪϪϩ,p ϪϩϩϪ F1e8 ϪϪϪϪϪϩ,p Ϫϩϩ * F1e9 ϪϪϪϪϪϩ,p Ϫϩ * V2.2.2 E4e1 ϩϩϪϪϪ ϩϩϩϪϩE4E1 Ϫ E4e2 ϪϪϪϪ * ϩ ϪϪϪE4E2 Ϫ E4e4 ϩϩϪϪϪ Ϫ Ϫ ϪϩϩE4E3 Ϫ E1e4 ϪϪϪϪϪϩ,p ϪϩϩϩϩE4E4 ϩ E1e5 ϪϪϪϪϪϩϩ,p ϪϪϪE4E5 Ϫ E4E6 Ϫ E4E7 * E1E3 ϩ E4E9 Ϫ E1E1 ϩϩ E1E2 ϩϩ

Note. See footnote to Table 1.

Somewhat surprisingly, each of the blastomeres of the DISCUSSION 16-cell-stage embryo contributes to the development of the red blood cells, although the vegetal-pole blastomeres Despite intense interest in the field of developmental tend to contribute a greater volume of tissue to the blood angiogenesis, the origins of vascular tissue in vertebrates islands. Sixty-four percent of D1.1, 60% of D1.2, 82% of have remained elusive. The purpose of this study was to D2.1, 50% of D2.2, 80% of V1.1, 50% of V1.2, 83% of provide a fate map for red blood cells as well as specific V2.1, and 80% of V2.2 injected blastomeres contributed structures of the vascular system of the 16- and 32-cell stage to this tissue (Table 1; Figs. 4G–4I). In no case was one Xenopus embryo with the goal of elucidating the origins of blastomere responsible for the entire lateral or dorsal- the vascular system and testing commonly held hypotheses ventral width of the blood islands in any given section, regarding the origins of these structures. Our study pro- suggesting that a mosaic of blastomeres is required for poses several basic conclusions. ventral blood island formation. Individual blastomeres First, the data presented in this study suggest that the gave rise to discrete patches of tissue in which, in some progenitor cells for vascular endothelium are located instances, many adjacent cells would exhibit RLDx sig- throughout cleavage stage embryos in X. laevis; every nal or, in other instances, many individual cells scattered blastomere in the 16-cell-stage as well as in the 32-cell- throughout the blood islands would display RLDx. When stage embryo gives rise to progeny that contributes to some examined at the 32-cell stage, red blood cell precursors part of the vasculature. The vegetal pole blastomeres, remained widespread throughout the embryo. However, however, at both the 16- and the 32-cell stages contribute to interestingly, while all of the animal-most tier of the a greater diversity of vascular structures and generally 16-cell-stage embryo gave rise to cells that would later provide a greater percentage of tissue to a given vascular express globin, at the 32-cell stage, only the vegetal tier structure than the animal pole blastomeres. It is interesting of these daughter cells consistently gave rise to globin- to note that progenitors for vasculature are significantly expressing cells, suggesting an asymmetric cell division more widespread throughout the embryo than other meso- (Table 2). dermal structures, for example, notochord and somites

(Moody, 1987a,b; Dale and Slack, 1987). The fate maps of dium. In the remaining cases in which a given blastomere Moody and Dale and Slack did not specifically address the produces progeny that contribute either to the myocardium vascular endothelial progenitors, but a ventral origin for (40%) or to the endocardium (15%), the dorsal blastomeres this cell type is generally assumed in the literature (e.g., see actually provide more progeny to both the myocardial and Lee et al., 1994; Drake et al., 1997). Although a lineage the endocardial lineages. Although the conclusions set analysis of vascular endothelial tissue was not the primary forth by Lee et al. (1994) are inconsistent with the results focus of the experiments by Rovainen (1991), he also reported here, this may be attributable to fact that their maintained that vascular structures arose from ventral study was performed in zebrafish slightly later, at the progenitors. However, this study did not identify individual 512-cell stage, and that it examined primarily superficial blastomeres at the time of injection nor was there an cells. attempt to perform systematic injections on all the blas- Virtually no restriction of this widespread potential to tomeres at a given stage, factors that may explain the produce vascular endothelial progeny between the 16-cell discrepancies between this study and that of Rovainen. stage and the 32-cell stage was observed, either in general or Second, even individual vascular structures consistently for specific vascular structures. Although these results are derived from a wide area of the embryo. For example, at the unexpected in light of the commonly held view that pro- 16-cell stage every blastomere contributed to both the genitors for vascular endothelial cells are localized to the aortic arches and the cardinal veins while at the 32-cell ventral or lateral splanchnic mesoderm, they are consistent stage a significant number of blastomeres contributed to with a number of other studies. Using a series of hetero- the aortic arches (75%) and cardinal veins (94%). It is topic transplants in the chick–quail system, Noden (1990) important to note, however, that certain blastomeres con- showed that even at later (neurula) stages the ability of sistently contribute a greater percentage of tissue to a given tissues to give rise to vasculature remains significantly structure, for example, D2.2 to the aortic arches or V2.1 to widespread with only head and notochord unable to do so. the cardinal veins (see Tables 1 and 2). For the remaining He concluded that much of the mesodermal tissue has the vascular structures examined, at the 16-cell-stage, 62% of potential to give rise to vascular endothelial cells. Other the blastomeres contributed to the endocardium and 75% studies demonstrating angiogenic potential of the somites to the dorsal aorta and lateral abdominal veins. Even at the as well as of the cephalic mesoderm, for example, support 32-cell stage a significant majority of the blastomeres these conclusions (Wilting et al., 1995a; Couly et al., 1995). produced progeny that populated the endocardium (56%), Our third conclusion is that although individual vascular dorsal aorta (75%), and lateral abdominal veins (75%). structures consistently derive from a wide area of the While such a result may be expected for vasculature extend- embryo, different blastomeres preferentially do give rise to ing the length of the embryo such as the dorsal aorta or different vascular structures. For example, the vegetal pole cardinal veins, it is somewhat surprising for more localized blastomeres (e.g., V2.1.1, V2.2.1, D2.2.1, D2.1.1) preferen- structures such as the endocardium. In zebrafish, Lee et al. tially contribute to the lateral abdominal veins, while the (1994) showed that while myocardial precursors are distrib- vegetal pole blastomeres on the ventral side (e.g., V2.1.2, uted throughout the marginal zone, endocardial precursors V2.2.2, V2.1.1, V2.2.1) give rise to progeny in the cardinal are restricted to the extreme ventral marginal cells. Our veins, and the dorsal blastomeres (e.g., D2.2.1, D2.2.2, results demonstrate that in 45% of the cases analyzed at D1.1.2) give rise to the aortic arches. Those blastomeres both the 16-cell stage and the 32-cell stage, the same that contribute with high frequency to a given structure blastomere gives rise to both myocardium and endocar- also generally tend to provide a greater percentage of tissue

FIG. 3. Colocalization of X-msr signal and RLDx. Following in situ hybridization with an X-msr probe, embryos were embedded in wax, transversely sectioned, and then analyzed for the presence of both X-msr and RLDx. Black arrowheads (A, D, and G) indicate X-msr signal in anterior cardinal veins, endocardium, and posterior cardinal veins, respectively; white arrowheads (B, E, and H) indicate the same location when viewed under fluorescence. Black arrows (C, F, and I) show overlay of X-msr signal with fluorescence in the identical location. (A, B, and C) Examples of X-msr staining in the anterior cardinal veins colocalizing with RLDx from blastomere D2.2. (A) X-msr signal in anterior cardinal vein, (B) fluorescence in the same section, and (C) an overlay of both images showing colocalization. (D, E, and F) Examples of X-msr staining in the endocardium colocalizing with RLDx from blastomere D1.1. (D) X-msr signal, (E) fluorescence in the same section, and (F) an overlay of both images. Insets represent an enlargement of the upper left region of the endocardium showing X-msr signal (C), fluorescence (D), and overlay (E). Arrowheads in the insets point to two cells showing both X-msr signal and fluorescence; directly to the right of the asterisk is depicted a cell that shows X-msr signal but does not show fluorescence colocalization. (G, H, and I) Examples of X-msr signal in the posterior cardinal vein colocalizing with RLDx signal from blastomere V2.2. (G) X-msr signal, (H) fluorescence in the same section, and (I) an overlay of both images. For A–I, dorsal is toward the top of the page. Magnification for A–C is 250ϫ, D–F is 400ϫ, GandHis200ϫ,andIis180ϫ. Note that the magnification of G and H is slightly greater than that of I. (J) Schematic indicating planes of section: I (A, B, C), II (D, E, F), and III (G, H, I). Diagram adapted from Nieuwkoop and Faber (1967). acv, anterior cardinal vein; endo, endocardium; fg, foregut; mb, midbrain; myo, myocardium; nc, notochord; ret, retina; som, somite; pcv, posterior cardinal vein.

Copyright © 1999 by Academic Press. All rights of reproduction in any form reserved. 364 Mills, Kruep, and Saha to that structure. There are, however, exceptions to this lineage. Although this is surprising in light of commonly generalization; certain blastomeres consistently and repeat- accepted views on ventral origins of blood cells (e.g., Kelley edly contribute less than 35% of the total tissue volume to et al., 1994; Walmsley et al., 1994; Hemmati-Brivanlou and a given structure, for example, V2.2 to the lateral abdominal Thomsen, 1995), more recently it has been shown that veins and D2.2 to the dorsal aorta. Consistent with the ventral blood island formation is considerably more com- results of Moody (1987a,b) and Dale and Slack (1987, for plex than previously thought, involving significant contri- other structures) and Rovainen (1991, for vasculature), no butions from both dorsal and ventral regions of the embryo single blastomere at the 16- or 32-cell stage is responsible (Tracey et al., 1998). Between the 16-cell stage and the for populating a given vascular structure. This tendency for 32-cell stage, there is evidence of an asymmetric division certain blastomeres to give rise to specific regions of the perhaps reflecting segregation of certain factors or cell–cell vasculature may be an early manifestation of an inherent interactions that begin to restrict the red blood cell lineage. heterogeneity in the vasculature which is later reflected on It is noteworthy that these animal-pole cells that no longer the genetic level (Baldwin, 1996). For example, cloche give rise to progeny that express globin are the cells whose mutants preferentially affect head vasculature (Liao et al., progeny are required to provide inductive signals required 1997) while ephrins distinguish between arterial and ve- for red blood cell development (Maeno et al., 1994). nous tissue (Wang et al., 1998). There was no evidence of Although not providing conclusive evidence for the he- asymmetric cell divisions with one of the daughter blas- mangioblast hypothesis, these data are generally consistent tomeres producing progeny for a specific vascular structure with this view except for those blastomeres in the animal and the other not. tier of the 32-cell-stage embryo that appear not to contrib- Fourth, and not unexpectedly, our study suggests that the ute to the blood cell lineage but nevertheless still give rise lineage of vascular structures is also influenced by more to vasculature. This suggests that at least for these cells, global trends of patterning. The more dorsal vessels, such as there must be alternative mechanisms for the origin of the dorsal aorta, arise from the more dorsal, anterior blas- vascular cells. The hemangioblast hypothesis has been the tomeres and the more ventral vessels, such as the lateral subject of considerable discussion. The requirement of abdominal veins, arise from the more ventral blastomeres. certain gene products for normal vascular development as This is also evident along the anterior–posterior axis of well as hematopoiesis (see Introduction) is certainly sugges- vascular structures. For example, in the structures that tive of a common precursor. It is formally possible, how- traverse the length of the organism, the anterior regions ever, that two cell types from different lineages express a generally derive from the more dorsal blastomeres and the certain gene because this gene is involved in mediating posterior regions from the more ventral; this was true even “field properties,” such as Pax6 for eye and lens develop- for the aortic arches. This is consistent with established ment (Cvekl and Piatagorsky, 1996). However, other lines patterns of tissue movements during gastrulation as well as of evidence involving experimental manipulation also lend the linkage of anterior with dorsal fates and ventral with support to the hemangioblast theory: the ability of posterior fates (see, for example, Green et al., 1997). VEGFR2-positive cells from early mesoderm to give rise to Fifth, our data suggest that the progenitors for red blood hematopoietic or endothelial cell types in a ligand- cells are far more widely distributed throughout the 16- and dependent manner (Eichmann et al., 1997), the existence of 32- cell-stage embryo, with significant contributions from endothelial precursors within the adult blood (Asahara et both dorsal and ventral blastomeres. Although the ventral, al., 1998), and the ability of avian QCE-6 cells to produce vegetal blastomeres contributed progeny most consistently myocardial, endocardial, and red blood cell phenotypes in at both stages, every blastomere at the 16-cell stage con- culture (Eisenberg and Markwald, 1997). On the other hand, tributed and all but a few blastomeres from the most animal Palis et al. (1995) have shown that primitive erythropoiesis tier of the 32-cell-stage embryo contributed to the red blood in extraembryonic mesoderm does not require a vascular

FIG. 4. Colocalization of X-msr or ␤-globin and RLDx. Following in situ hybridization, embryos were embedded in wax and transversely sectioned and then analyzed for the presence of either X-msr or ␤-globin and RLDx. Black arrowheads in A, D, and G indicate in situ hybridization signal for lateral abdominal veins, dorsal aorta, and ventral blood islands; white arrowheads in B, E, and H indicate the same location viewed under fluorescence. Arrows in C, F, and I show overlay of in situ hybridization signal with fluorescence in same region. (A, B, and C) Examples of X-msr staining in the lateral abdominal veins colocalizing with RLDx from blastomere V2.2. (A) X-msr signal, (B) fluorescence in the same section, and (C) an overlay of both images showing colocalization. (D, E, and F) Examples of X-msr signal colocalizing with RLDx from blastomere V2.1. (D) X-msr staining in the dorsal aorta, which lies ventral to the notochord and hypochord, (E) fluorescence in the same section, and (F) an overlay of both images. (G, H, and I) Examples of ␤-globin signal in the ventral blood islands colocalizing with RLDx from blastomere D2.1. (G) ␤-Globin signal, (H) fluorescence in the same section, and (I) an overlay of both images. Dorsal is toward the top of the page. Magnification for A–C and G–I is 200ϫ, D–F is 250ϫ. (J) Schematic indicating planes of section: I (A, B, C), II (D, E, F), and III (G, H, I). Diagram adapted from Nieuwkoop and Faber (1967). da, dorsal aorta; hg, hindgut; lav, lateral abdominal vein; nc, notochord; som, somite; vbi, ventral blood island.

FIG. 5. Schematic summary of fate map for (A) the 16-cell- and (B) the 32-cell-stage embryos. The embryos schematically depict the progeny of each of the blastomeres of the 16- and 32-cell-stage embryos. The percentage of injected embryos for a given blastomere that contributed to a speciﬁc tissue is indicated as follows: Structures denoted in large bold red letters indicate that 75–100% of the injected blastomeres contributed to that structure; structures shown in large blue letters denote that 50–74% of the injected embryos contribute progeny to that structure; structures shown in small black letters indicate that 25–49% of the injected blastomeres contributed progeny to the structure.

network or yolk sac endoderm. Pardanaud et al. (1996), and the extent of the contribution, it is important to note using orthotopic and heterotopic grafts, demonstrated that that our fate maps reflect probabilities of the distribution of endothelial precursors from paraxial mesoderm never asso- the progeny of given blastomeres. As demonstrated in ciated with blood cell formation. Liao et al. (1997) also previous fate maps for other tissues (Moody, 1987a,b; Dale suggest at least two endothelial lineages, a trunk and tail and Slack, 1987), it is evident that lineage is not a simple lineage that is associated with red blood cells and a head determinant of vascular endothelial or red blood cell fate in endothelial lineage that is not. Thus there is considerable X. laevis. This indeterminacy is attributable to the fact that support for the view that while certain vascular endothelial the conclusive fate of a specific blastomere is dependent cells arise from hemangioblasts, there is also an alternative upon intricate cellular interactions and tissue rearrange- pathway not involving erythropoiesis (Palis et al., 1995; ments that are accompanied by a high degree of develop- Pardanaud et al., 1996). Our study supports this view; mental plasticity. However, the goal of this study was not however, to resolve this issue it is necessary to identify to address the role of lineage per se in cell determination when, after the 32-cell stage, the divergence of these two but rather to examine the location of vascular endothelial lineages occurs. Moreover, in order to obtain a complete picture, it will also be essential to examine the lineage of and red blood cell precursors in the 16- and 32- cell-stage related cell types such as lymphatic endothelial cells (Wilt- embryo, stages at which regional specification has already ing et al., 1996, 1998), other hematopoietic cell types commenced and at which the progenitors of other mesoder- (Dieterlen-Lievre et al., 1997; Dieterlen-Lievre, 1998), and mal derivatives are somewhat more localized. With a macrophages (Cuadros and Navascues, 1998). knowledge of the specific spatial addresses of the vascular Finally, although there are clear trends for the fates of precursors in the early amphibian embryo, we are now in a each blastomere with respect to vascular endothelial cells position to manipulate the development of the system at and red blood cells, in terms of both the percentage of the earliest point in its determination with a full under- injected blastomeres that contribute to a given structure standing of the normal fate of these cells.

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F., and Maniatis, T. (1989). “Molecular Received for publication October 13, 1998 Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory Revised February 4, 1999 Press, Cold Spring Harbor, NY. Accepted February 15, 1999