Intraembryonic Hematopoietic Cell Migration During Vertebrate Development H

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 10713-10717, November 1995 Developmental Biology

Intraembryonic hematopoietic cell migration during vertebrate development H. WILLIAM DETRICH TI1*t, MARK W. KIERANt, FUNG YEE CHANt#, LAUREN M. BARONEtt, KAREN YEEt, JON A. RUNDSTADLERt, STEVEN PRAT[t, DAVID RANSOMtt, AND LEONARD I. ZONt#§ *Department of Biology, Northeastern University, Boston, MA 02115; and tHoward Hughes Medical Institute, and tDivision of Hematology/Oncology, Children's Hospital and Dana-Farber Cancer Institute, Department of Pediatrics, Harvard Medical School, Boston, MA 02115 Communicated by Elizabeth S. Russell, The Jackson Laboratory, Bar Harbor, ME, June 30, 1995

ABSTRACT Vertebrate hematopoietic stem cells are de- species studied thus far, GATA-1 levels increase and GATA-2 rived from ventral mesoderm, which is postulated to migrate levels decrease during erythroid differentiation (17). to both extra- and intraembryonic positions during gastrula The existence of distinct ventral (yolk sac) and dorsal and neurula stages. Extraembryonic migration has previously (intraembryonic) hematopoietic compartments in vertebrate been documented, but the origin and migration of intraem- embryos has been known for many years (18-24), but the bryonic hematopoietic cells have not been visualized. The origin and migration pathway of the dorsal compartment and zebrafish and most other teleosts do not form yolk sac blood its relationship to the ventral compartment remain to be islands during early embryogenesis, but instead hematopoi- determined. To address this issue, we have studied blood esis occurs solely in a dorsal location known as the interme- formation in the zebrafish, Danio rerio, which utilizes only a diate cell mass (IM) of Oellacher. In this report, we have dorsal compartment during embryogenesis. Using GATA-1 isolated cDNAs encoding zebrafish homologs of the hemato- and GATA-21 as early markers of hematopoietic tissue (14, poietic transcription factors GATA-1 and GATA-2 and have 17), we have demonstrated that the dorsal population of cells used these markers to determine that the IM is formed from containing these markers is derived from posterior-lateral mesodermal cells in a posterior-lateral position on the yolk mesoderm at the gastrula stage, and we have delineated the syncytial layer of the gastrula yolk sac. Surprisingly, cells of migration of the marked cells during development to the dorsal the IM then migrate anteriorly through most of the body mesentery. These results suggest that similar migration path- length prior to the onset ofactive circulation and exit onto the ways are used in vertebrates for the formation of both ventral yolk sac. These findings support a hypothesis in which the and dorsal hematopoietic populations. hematopoietic program of vertebrates is established by variations in homologous migration pathways of extra- and in- METHODS traembryonic progenitors. Zebrafish Maintenance. Wild-type zebrafish stocks were obtained from Ekk Will Waterlife Resources (Gibsonton, FL). In 1872, Joseph Oellacher demonstrated that embryonic he- Zebrafish matings were performed according to standard matopoiesis in most teleosts occurs in a dorsal intraembryonic protocols (25). Wild-type embryos from crosses of spadetail region of the tail bud called the intermediate cell mass (IM) heterozygous mutants (spadetail provided by C. Kimmel, Uni- (1). The intraembryonic site of hematopoiesis in bony fishes versity of Oregon, Eugene) were occasionally used. has perplexed developmental biologists, because at an analo- Isolation of cDNA Clones Encoding Zebrafish GATA-1 and gous developmental stage, all other vertebrate species that GATA-2. ZG1 and ZG2 were isolated from 36-h Agtll and have been examined form blood on the extraembryonic yolk neurula AZapII cDNA libraries, respectively, by screening at sac (1-13). In 1885, Wenckebach (12) hypothesized that the IM reduced stringency (26) with Xenopus GATA-1 (XG1) cDNA. was formed by the convergent migration of two bilateral From 5 x 105 plaques per library, one GATA-1 cDNA and 23 populations of hematopoietic progenitors; nevertheless, care- GATA-2 cDNAs were isolated. Phage cDNA inserts were ful studies in the 20th century have failed to define the subcloned into Bluescript KS1I- (Stratagene) and sequenced developmental origin of the IM (2-11). by the dideoxynucleotide chain-termination method (26, 27). Vertebrate embryonic hematopoiesis involves critical tran- Whole Embryo Staining for Globin Expression. o-Dianisidine scriptional regulation of coordinately expressed genes such as staining was used to study the expression of globin (28). Decho- the globins, heme biosynthetic proteins, and cell-surface re- rionated (nonfixed) embryos were stained for 15 min in the dark ceptors. Functionally defined GATA motifs, which are recog- in o-dianisidine (0.6 mg/ml), 0.01 M sodium acetate (pH 4.5), nized by GATA-binding transcription factors, are present in 0.65% H202, and 40% (vol/vol) ethanol. Stained embryos were the promoters and enhancers of many hematopoietic-specific cleared with benzyl benzoate/benzyl alcohol (2:1, vol/vol) and genes (14). In mice, disruption of the Gatal or Gata2 gene examined by differential interference contrast microscopy. leads to severe hematopoietic defects, demonstrating a re- Whole Embryo in Situ Analysis. In situ hybridization was quirement for these factors for normal erythroid maturation performed by a modification of the method of Harland (29). (15, 16). GATA-1 and GATA-2 have recently been used in After digestion with proteinase K, treatment with acetic Xenopus as markers of hematopoietic mesoderm induction anhydride, and prehybridization, embryos were incubated with (17). GATA-1 and GATA-2 are initially expressed during digoxigenin-labeled antisense or sense RNA probes (1 ,ug/ml gastrulation, and GATA-1 expression is restricted to hemato- in hybridization buffer) for 14-16 h at 60°C. The embryos were poietic cells during embryogenesis. GATA-2 is expressed in early ventral ectoderm and hematopoietic cells and later is Abbreviations: IM, intermediate cell mass; DM, dorsal mesentery. expressed in the central nervous system. In each vertebrate §To whom reprint requests should be addressed at: Division of Hematology/Oncology, Children's Hospital, 300 Longwood Avenue, Enders 650, Boston, MA 02115. The publication costs of this article were defrayed in part by page charge 1The sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" in GenBank data base [accession nos. U18311 (GATA-1) and U18312 accordance with 18 U.S.C. §1734 solely to indicate this fact. (GATA-2)]. 10713 Downloaded by guest on September 27, 2021 10714 Developmental Biology: Detrich et al. Proc. Natl. Acad. Sci. USA 92 (1995)

treated with RNase and washed to a stringency of 0.2x SSC/ from an ancestral GATA-2 molecule before or during the 0.3% CHAPS (60°C). Transcripts were detected by incubation evolution of fish and that the structure of GATA-1 has with alkaline phosphatase-conjugated anti-digoxigenin Fab frag- diverged from that of GATA-2 in more advanced vertebrates. ments (Boehringer Mannheim), followed by development with GATA-1 Expression Delineates the Migration of Hemato- nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate poietic Cells During Development. The erythroid cells of the (Promega). Sense control transcripts produced no signal. Some zebrafish IM are easily distinguished by their small size, oval embryos were dehydrated, infiltrated with JB4 resin (Poly- shape, slightly condensed chromatin (3), hemoglobin expres- science), and sectioned (5 ,um). sion based on o-dianisidine staining (Fig. 2 Q and R) (3, 4), and GATA-1 mRNA expression (Fig. 2H and data not shown). Based on whole embryo in situ analysis, GATA-1 expression is RESULTS not detected in any other embryonic tissue. Therefore, as in the Cloning of the Zebrafish GATA-1 and GATA-2 cDNAs. development of other vertebrates (17, 32), GATA-1 mRNA cDNA clones encoding GATA-1 and GATA-2 were obtained expression is restricted to hematopoietic cells during zebrafish by screening zebrafish cDNA libraries with the zinc finger embryogenesis. GATA-1 mRNA is initially detected at the region of Xenopus GATA-1 cDNA (Fig. 1) (30). Sequence 2-somite stage in putative progenitors that reside in two stripes analysis of the encoded polypeptides demonstrated that the flanking the paraxial mesoderm of the posterior embryo and primary structure of zebrafish GATA-1 is very similar to that that contact the yolk syncytial layer [Fig. 2A and B (arrows)]. of zebrafish GATA-2, particularly in the zinc finger region. This embryonic region contains lateral mesoderm that some The extreme N termini of the two zebrafish transcription investigators consider extraembryonic (33-35) (Y. Kunz, per- factors are significantly more homologous to each other than sonal communication) and others regard as embryonic (W. are the N termini of Xenopus GATA-1 and GATA-2 (Fig. 1; Ballard, personal communication). The two cell masses that XG1 and XG2, respectively). This suggests that GATA-1 arose express GATA-1 converge medially between the 2- and 5-somite stages (Fig. 2 C and D), meet anteriorly at the 1 50 18-somite stage, and fuse completely by the 24-somite stage ZG2 EVAADQSRW MAHHHA QGOHSHHHGL TH EPMAP LLPPDEVDV (Fig. 2 E-H). Examination of the GATA-1-expressing cells ZGl MENSSEPSRW VS... PG Sp ...V . ..rTDSGL LPPVDVDEP

XGI1 . N DY.. .T qT Q P .... 4. ESGL ASTSE.DSOE from the 2- to 18-somite stages by differential interference XG2 MEVATDQPRW MAH.NAV G QH SHHPGL AHN MEP.TQ LLPPDEVDVM contrast microscopy demonstrated that they are large, round 51 100 ZG2 LNHLDSQGN .Y SNS... ARVSYGQAHA RLTGSQVCRP HLIHSPGIPW cells with prominent nuclei that differentiate to the smaller ZGI YSSSETDLLP SY STSVQS SSYRHSPV RQVYSSQSIL GNIQWL.... erythroblast stage as the cells meet at midline. These eryth- XG1 LYGLGGES G GGAVSS GFRSPV FQTFPVGR.LHWP.... XG2 FNHLDSQGN .YIANSAHA a RVSYSQAHA RLTGSQMCRP HLLHSPGLPW roblasts are distinct from larger interspersed cells of the IM 101 150 that differentiate into endothelial cells between 18 and 24 ZG2 LDSGKAALSA A.HHNAWAVS HFSKPGLHPA ... SAAY CS SSSSTAPVSS somites (3). At 23 h [ust before the prim 5 stage (25)], ZG1 ...... DNSAGH SLN.SPYNPT STVWSSSFP KTPLHSHTST XG1 ...... ETSAGI PSNLTAYGRS TGTLSF A ASALGPITSP GATA-1-expressing cells of the IM migrate anteriorly (Fig. 2 XG2 LESGKTALSA AHHHNPWTVS PFGKAPLHPA ARGGSLY GT GSSACP.... I and J). By 24 h, the cells have reached the midtrunk region 151 200 and to exit onto the sac 2 ZG2 LTSATHSSPH PLYNLPPTPP KDVSPDPGPS SPTSTTARMD EKE KYQVS (Fig. 2K, arrow) appear yolk (Fig. ZG1 SIYQNTATPS FTSPKEGFPS PSR...... D GK RLQ.. L-N) between the ectoderm and the yolk surface (sections not XG1 ...... PLY.. SASS FLLG..... SA PPA....E RE KFL.. XG2 ...... SSSHSSPH .LFGFPPTPP KDVSPDPGPA SPPS.SSRLE DK KYQMS shown). On the yolk sac, the erythroid cells mature, charac- 201 _ 250 terized by increased nuclear condensation and a slightly more ZG2 IADG C SPLRGS.... LAMSAQTPST HHPIPTYP.. .TYSLRAPHD elliptical cell shape. The migration of hematopoietic cells ZGI . . E L SPMSGSGSSF LSSATGGV YGPSPHMLSP YGSYMSTSQD XG1 .ET A SP ... TSDL PLEPRSPSI LQV...... GYIGGGGQE initiates before an active circulation is established at 24 h (6). XG2 LSE G SPLRSS.... LAPMGTQCST HHPIPTYP.. SY.VPAAHD Between 25 and 29 h, the common cardinal veins and ducts of 251 300 Cuvier form (Fig. 20), and the blood cells appear to be ZG2 YGGGLFHPGA LLSGSASSFT PKCKSKTRSC SEFCTEG EC VNCGAT ZG1 YSSAALYSTG GPWMSPSSYS PKLRNKMRLS P ... PEA EC VNCGAITArPLl channeled toward the heart (Fig. 2P, arrow). The anterior XG1 FS ..... LFQ S .. TED REC NCGAT VrP1I XG2 YSSGLFHPGLLGGPASSFTS PKQRSKSRSC S....EGCVNCGAT accumulation of blood cells on the yolk sac has also been visualized video of zebrafish em- 301 350 by time-lapse microscopy ZG2 HY ACGLY QMRPL C bryos (C. Kimmel, personal communication; M. Thompson ZG1 HY AC LY GQNRPL LVS S XG1 HY ACOLY GQNRPL VS S S and L.I.Z., unpublished observations). XG2 NPLGLYNCR GATA-2 Is Expressed in an Early Hematopoietic Progenitor 351 400 Population. GATA-2 expression during development is simi- ZG2 rTTLWRGN VNACG L LT IQ K ZG1 rTTLWRRAS VCNACG Y L LTMK IQ lar, although not identical, to that of GATA-1. GATA-2 XG1 rITTLWRRAS NACGL Y L LT IQ R XG2 rTLWRR NACGS L mRNA is initially detected in the ventral ectoderm at 75% epiboly (Fig. 3A), and high levels are expressed at the bound- 401 450 ZG2 ELSKCMQDKT SPFGTASA . ASHMPHM PPFSHS ary of the embryo and in the yolk syncytial layer at 90% epiboly ZG1 KRSFqRSGEGFENSEV YPDMSHMAPP DEHVGAYSI PGPLLSY ..... P XG1 K.WQLDNPF EPPKAGVEEP SPYPFGPI4 HGQMPPM . INPP (Fig. 3 B and C). By the 2- to 5-somite stages, GATA-2 XG2 KEJGSECFE ELSRCMQEKS SPFS. AAPAI. ASHMAPM APFSHS expression is observed in cells that border the anterior (Fig. 451 480 3D, closed arrow) and posterior (open arrow) embryo, includ- ZG2 PTPTPIHP.. ..TFSHPHHS GRSPAWAEPH ZG1 PTST.LHSST TLPYTHHPNFI GMMPTLV... ing the presumptive hematopoietic progenitors. From the 5- to XG1 QSPR.ISHSA PAVSYRQAA84 GVTPP..... XG2 QTPTPIHPSS SLSFGHPHSW TAMG... 20-somite stages, these progenitors continue to express GATA-2 mRNA (data not shown). Large cells in the posterior FIG. 1. Primary sequences of zebrafish GATA-I and GATA-2. For IM express abundant GATA-2 mRNA but do not express purposes of comparison, zebrafish GATA-1 (ZG1) and GATA-2 GATA-1 (compare Fig. 2G, open arrow with Fig. 3E, open (ZG2) have been aligned with Xenopus GATA-1 (XG1) and GATA-2 arrow). Because of their blast-like morphology (3) and the (XG2) (30). Periods denote gaps introduced to establish maximal absence of GATA-1 expression, this cell population may sequence similarity. Boxed residues are shared by all four GATA- represent undifferentiated embryonic or larval/adult stem binding proteins, and the dashed lines indicate the two zinc fingers. cells. After the 20-somite stage, the level of GATA-2 mRNA Based on GAP analysis (Genetics Computer Group format), ZG1 is not 45.2% identical to ZG2 and 48.3% identical to XG1, while ZG2 and in the anterior cells of the IM decreases substantially (data XG2 are 78.2% identical. The longest ZG2 cDNA obtained lacked the shown), consistent with its declining expression in embryonic initiator codon and started with the second codon; we infer that erythroid cells after blood island formation in higher verte- methionine is the first amino acid based on homology to all other brates (30). The intense GATA-2 expression throughout the vertebrate GATA-2 polypeptide sequences (30). IM (Fig. 3E) may be due to both hematopoietic and vasculo- Downloaded by guest on September 27, 2021 Developmental Biology: Detrich et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10715 C D

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FIG. 2. Expression of GATA-1 and globin during zebrafish embryogenesis. In situ hybridization was performed with fixed zebrafish embryos at the following stages: (A and B) Two somites. (C and D) Five somites. (E and F) Eighteen somites. (G and H) Twenty-three hours (before prim 5 stage). (I) Twenty-four hours (prim 5). (J) Twenty-five hours. (K) Twenty-six hours. (L-P) Twenty-six to 29 h. (B, D, F, and H) Cross-sections through the posterior region or tail bud (IM) of embryos corresponding to (A, C, E, and G). For each section, e denotes the embryo and y indicates the yolk. The arrows in B show GATA-1-expressing progenitors that reside in contact with the yolk syncytial layer. The closed arrow in G indicates the posterior boundary of GATA-1 expression in the IM. The open arrow in G points to undifferentiated, GATA-2-expressing progenitors in the posterior IM (see Fig. 3E). The arrows in J and K show hematopoietic progenitors migrating anteriorly and onto the yolk sac. Erythroid cells later enter the heart (P, arrow). [M, 0, and P are normal sibs from a cross of two spadetail (31) heterozygotes.] (Q and R) o-Dianisidine staining of 24-h wild-type zebrafish embryos. The brown color indicates erythroid cells containing hemoglobin in the IM region. Downloaded by guest on September 27, 2021 10716 Developmental Biology: Detrich et al. Proc. Natl. Acad. Sci. USA 92 (1995)

Y A are derived from cells that occupy the rim of the posterior embryo proper, similar to those that give rise to the IM in zebrafish (10, 11, 36). Preliminary in situ analyses in angelfish embryos demonstrate GATA-1 expression in two bilateral cell populations on the yolk sac (data not shown). Therefore, based on GATA-1 expression detected during gastrulation, yolk sac blood islands and the IM are likely to derive from similarly localized stem cell populations. The spatial distribution of hematopoietic stem cells in fish D embryos may be established by variations in morphogenetic movements. In embryos, such as Fundulus, that have extended epiboly and rapid gastrulation, hematopoietic progenitors remain at the lateral border of the embryo proper and subsequently migrate onto the yolk sac to form blood islands. In embryos characterized by rapid epiboly and slower gastrulation, such as the zebrafish, IM formation predominates, FIG. 3. Expression of GATA-2 mRNA in zebrafish. (A) Seventy- apparently because hematopoietic cells are subjected to con- five percent epiboly. Open arrows represent the border of the embryo, vergent mesodermal migration. Alternatively, specific induc- and closed black arrows indicate expression in ventral ectoderm. (B ers or chemotactic molecules may regulate the localization of and C) Ninety percent epiboly. GATA-2 is expressed within the blood cell populations. advancing yolk syncytial layer, with higher levels at the embryonic boundary (white arrows). (D) Five somites. A "baseball" pattern of Our studies on the zebrafish demonstrate a propensity for expression encompasses the anterior (solid arrow) and posterior (open fish hematopoietic progenitors, which may constitute the arrow) regions of the embryo. (E) Twenty somites. Note the expression embryonic or postembryonic programs, to migrate to the throughout the IM, including the posterior undifferentiated progen- dorsal mesentery (DM). This process in teleosts, which we have itors (open arrow). The solid arrow indicates the posterior boundary directly visualized using antisense RNAs for the DNA-binding of GATA-1 expression in the IM. proteins GATA-1 and GATA-2 as cell markers, is remarkably similar to proposed hematopoietic cell migration pathways in genic transcription. GATA-2 has been implicated as an endo- other vertebrates. Reciprocal transplantations between cyto- thelial transcription factor involved in the regulation of pre- genetically marked amphibians (21) have demonstrated ante- pro-endothelin and the cell adhesion molecule VCAM-1 (14). rior migration of hematopoietic progenitors from a posterior Zebrafish GATA-2 mRNA is also detected in discrete neural region surrounding the pronephric duct to the dorsal mesen- structures including the hindbrain-midbrain border and indi- teric region including the ducts of Cuvier, dorsal aorta, and vidual spinal neurons, similar to higher species (17). pronephros. Intraembryonic hematopoiesis (including the DM region) has also been documented in avian development using DISCUSSION chicken-quail chimeras (24) and in mammalian development using early progenitor assays (22, 23). The IM-forming teleosts have been regarded as exceptions to Migration may have a prominent role in the induction of the the general rule ofyolk sac hematopoiesis in vertebrates. Some hematopoietic program by establishing the relative distribu- fish, such as killifish (Fundulus spp.) (10, 11, 36), angelfish tion of extra- and intraembryonic hematopoietic stem cells. (Pterophylum scalare) (2), and chondrichthyans (1, 3, 37) form Moore and Metcalf (38) demonstrated that removal of the blood predominantly on the yolk sac. Fundulus blood islands murine yolk sac prior to appreciable blood island formation Zebraf ish Killifish Rana Chicken Mouse

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LATE Vt DM

FIG. 4. Unifying hypothesis for hematopoietic cell migration during vertebrate embryogenesis. (Upper) Early stage. Hematopoietic stem cells occupy a ventral or lateral position of the posterior embryo (E) proper and either migrate to the yolk (Y) sac or to a dorsal, intraembryonic position. In the zebrafish, ventral mesoderm migrates medially to a position adjacent to the pronephric duct, but in the killifish, it migrates laterally onto the yolk sac. In amphibians (i.e., Rana), the embryonic ventral blood islands are v-shaped, and dorsal progenitors are located adjacent to the pronephros. In birds, the lateral progenitors are initially detected in the area opaca; in mammals, they reside at the boundary of the intra- and extraembryonic tissues during late gastrula stages. (Lower) Late stage. Yolk sac hematopoietic cells enter circulation, whereas dorsal hematopoietic cells migrate into a dorsal mesenteric (DM) region. From the latter position, the dorsal cells either migrate to the yolk sac, invade the adjacent vasculature to enter the circulation to the heart (H), remain in situ, or migrate to the thymus (T) to form T cells. Sites such as the human fetal liver are colonized by circulating stem cells. Downloaded by guest on September 27, 2021 Developmental Biology: Detrich et al. Proc. Natl. Acad. Sci. USA 92 (1995) 10717 (day 7) led to a marked deficiency of fetal liver hematopoiesis, 3. Al-Adhami, M. A. & Kunz, Y. W. (1977) Dev. Growth Differ. 19, suggesting an early medial migration of yolk sac hematopoietic 171-179. cells to the embryo. It appears that the dorsal hematopoietic 4. Colle-Vandevelde, A. (1963) Nature (London) 198, 1223. population mostly contributes to fetal and adult hematopoiesis 5. Iuchi, I. (1973) J. Exp. Zool. 184, 383-396. in higher vertebrates, but in lower vertebrates, embryonic 6. Reib, J. (1973) Ann. Embryol. Morphog. 6, 43-54. hematopoietic progenitors are 7. Zapata, A. (1983) Bull. Inst. Pasteur (Paris) 81, 165-186. also found in the DM. For 8. John, C. C. (1931) Clupea 110, 112-119. instance, in Rana catesbeiana, it is likely that the larval/adult 9. Ruckert, C. B. & Mollier, S. (1906) Die erste Entstehung der progenitors of the DM colonize the larval liver, while the Gefasse und de Blutes bei Wirbeltieren, Handbuch der Ver- embryonic progenitors of the DM colonize the larval prone- gleichenden und Experimentellen Entwickelungslehre der Wir- phros (39). Such migration, which appears independent of beltiere (Hertwig, Germany), Vol. 1, pp. 1125-1153. active circulatory flow, may be mediated by a selective, direc- 10. Stockard, C. R. (1915) Am. J. Anat. 18, 227-327. tional gradient of cell adhesion with the developing vasculature 11. Stockard, C. (1915) Am. J. Anat. 18, 525-594. and pronephric duct or by long- or short-range chemotaxis. In 12. Wenckebach, K. F. (1885) J. Anat. Physiol. 19, 231-236. addition, the migration of extraembryonic mesoderm to form 13. Ziegler, H. E. (1887) Arch. Mikrosk. Anat. Entwicklungsmech. 30, hematopoietic cells in the dorsal mesentery is reminiscent of 596-665. the 14. Orkin, S. H. (1992) Blood 80, 575-581. migration of extraembryonic germ cell progenitors to an 15. Tsai, F.-Y., Keller, G., Kuo, F. C., Weiss, M., Chen, J., Rosen- intraembryonic compartment (40-42). blatt, M., Alt, F. W. & Orkin, S. H. (1994) Nature (London) 371, In 1963, Colle-Vandevelde concluded that "a better knowl- 221-226. edge of the origin of those blood cells might enable us to 16. Pevny, L., Simon, M. C., Robertson, E., Klein, W. H., Tsai, S. F., establish a connection between the classical 'dorsal' origin of D'Agati, V., Orkin, S. H. & Costantini, F. (1991) Nature (Lon- blood in the fish and its 'ventral' origin in other vertebrates" don) 349, 257-260. (4). Clearly, each vertebrate species has unique features of 17. Kelley, C., Yee, K., Harland, R. & Zon, L. (1994) Dev. Biol. 165, embryonic hematopoiesis, and it is difficult to relate the 193-205. extraembryonic membranes of amniotes to the yolk sac tissues 18. Iuchi, I. (1985) Zool. Sci. 2, 11-23. of fish. Nevertheless, a unifying hypothesis (Fig. 4), based on 19. Romanoff, A. L. (1960) in The Avian Embryo, ed. Romanoff, A. L. (Macmillan, New York), pp. 569-678. previous studies and our studies of the expression of GATA-1 20. Ingram, V. M. (1972) Nature (London) 235, 338-339. and GATA-2 in fish, postulates that homologous migration 21. Turpen, J. B. & Knudson, C. M. (1982) Dev. Biol. 89, 138-151. pathways play a central role in establishing the vertebrate 22. Medvinsky, A. L., Samoylina, N. L., Muller, A. M. & Dzierzak, hematopoietic program. Two primordial pathways are exem- E. A. (1993) Nature (London) 364, 64-67. plified by migration to the yolk sac in killifish and by intraem- 23. Godin, I. E., Garcia-Porrere, J. A., Coutinho, A., Dieterlen- bryonic migration in the zebrafish. Blood is derived from Lievre, F. & Marcos, M. A. R. (1993) Nature (London) 364, ventral or lateral mesoderm of the posterior embryo. This 67-70. mesoderm either travels further laterally and colonizes the 24. Dieterlen, L. F. & Martin, C. (1981) Dev. Biol. 88, 180-191. yolk sac as blood islands (19, 43) or migrates medially. In- 25. Westerfield, M. (1989) The Zebrafish Book: A Guide for the traembryonic cells Laboratory Use of Zebrafish (Brachydanio rerio) (Univ. Oregon then move anteriorly into the DM region. Press, Eugene, OR). Some DM hematopoietic cells populate the yolk sac, some 26. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular enter the vasculature into circulation, and others move to the Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, thymus and form T cells. From the yolk sac, all hematopoietic Plainview, NY), 2nd Ed. cells travel to the ducts of Cuvier to enter the embryonic 27. Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448. circulation. Thus, we postulate that the yolk sac receives two 28. Iuchi, I. & Yamamoto, M. (1983) J. Exp. Zool. 226, 409-417. waves of hematopoietic stem cells during development: cells 29. Harland, R. M. (1991) Methods Cell Biol. 36, 675-685. from lateral extraembryonic mesoderm that form embryonic 30. Zon, L. I., Mather, C., Burgess, S., Bolce, M., Harland, R. & blood islands and some later larval/adult colonies (44) and Orkin, S. H. (1991) Proc. Natl. Acad. Sci. USA 88, 10642-10646. cells from dorsal intraembryonic mesoderm that form pre- 31. Kimmel, C. B., Kane, D. A., Walker, C., Warga, R. M. & Roth- man, M. B. (1989) Nature (London) 337, 358-362. dominantly larval/adult hematopoietic cells (24). In summary, 32. Orkin, S. H. (1990) Cell 63, 665-672. our studies indicate that the hematopoietic program, including 33. Nelsen, 0. E. (1953) in Comparative Embryology of the Verte- homologous transcription factors and migration pathways, is brates, ed. Nelsen, 0. E. (Blakiston, New York), pp. 436-441. conserved in vertebrate evolution. 34. Fioroni, P. (1987) in Embryologie, ed. Fioroni, P. (Springer, Berlin), Vol. 183, pp. 230-231. The zebrafish cDNA libraries were provided by K. Zinn (California 35. Trinkaus, J. P. (1993) J. Exp. Zool. 265, 258-284. Institute of Technology) and R. Riggleman (University of Utah School 36. Stockard, C. R. (1915) Anat. Rec. 9, 124-127. of Medicine). We thank Y. Kunz, A. Colle-Vandevelde, W. Ballard, J. 37. Swaen, A. & Brachet, A. (1901) Arch. Biol. 18, 73-190. Trinkaus, J. Turpen, D. Stainier, and F. Dieterlen-Lievre for their 38. Moore, M. A. & Metcalf, D. (1970) Br. J. Haematol. 18,279-296. helpful discussions. We thank S. Lux, S. Orkin, M. Thompson, L. 39. Broyles, R. H. (1981) in Changes in the Blood DuringAmphibian Schneider, and C. Kimmel for critically reviewing the manuscript. Metamorphosis, eds. Gilbert, L. I. & Frieden, E. (Plenum, New L.I.Z. is an Assistant Investigator of the Howard Hughes Medical York), pp. 461-490. Institute. H.W.D. was supported by a U.S. Public Health Service 40. Rosenquist, G. C. (1966) Contrib. Embryol. Carnegie Inst. 38, National Research Service Award Senior Fellowship (HL09061). This 71-110. work was supported by grants from the National Institutes of Health 41. Gardner, R. L., Lyon, M. F., Evans, E. P. & Burtenshaw, M. D. (HL48801; L.I.Z.), National Science' Foundation (OPP-9120311; (1985) J. -Embryol. Exp. Morphol. 88, 349-363. H.W.D.), and Medical Research Council of Canada (M.W.K.). 42. Bianchi, D. W., Wilkins-Haug, L. E., Enders, A. C. & Hay, E. D. (1993) Am. J. Med. Genet. 46, 542-550. 1. Oellacher, J. (1872) Z. Wiss. Zool. 23, 373-421. 43. Tavassoli, M. (1991) Blood Cells 1, 269-281. 2. Al-Adhami, M. A. & Kunz, Y. W. (1976) Wilhelm RouxArch. 179, 44. Toles, J. F., Chui, D. H. K., Belbeck, L. W., Starr, E. & Barker, 393-401. J. E. (1989) Proc. Natl. Acad. Sci. USA 86, 7456-7459. Downloaded by guest on September 27, 2021