Ventral Blood Island Research Articles

Cloning and expression pattern of the Xenopus erythropoietin receptor

Cytokine signaling plays an important role in the survival and differentiation of vertebrate hematopoietic cells. In red blood cells, erythropoietin is a key component of the differentiation program and maintains the homeostasis of the erythroid compartment. In the adult, anemia stimulates high levels of circulating erythropoietin that drives erythropoiesis to restore normal levels of red blood cells in circulation. Erythropoietin activates the erythropoietin receptor on immature red blood cell precursors to promote their survival and differentiation. Although extensively studied in mammalian systems, a complete understanding of the function of the erythropoietin receptor during primitive erythropoiesis has been lacking. To address this problem, we have cloned the Xenopus laevis erythropoietin receptor in order to further understand the development of primitive erythropoiesis. The amphibian erythropoietin receptor shares 33% amino acid sequence identity with the mammalian erythropoietin receptors and contains the conserved extracellular ligand binding and fibronectin domains, the WSXWS motif common to cytokine receptors, and several tyrosine phosphorylation sites located on the intracellular domain of the receptor. Expression of the erythropoietin receptor is first detected by in situ hybridization in the ventral blood island during tailbud stages.

Developmental hematopoiesis from fly to human

Developmental hematopoiesis is an exciting multi-disciplinary field at the crossroads of developmental biology, hematopoiesis, stem cell biology and genetics. This field began nearly a century ago with detailed observations on the formation of the first blood cells, the primitive erythrocytes, in

Zebrafish KLF4 Is Essential for Primitive Haematopoiesis.

Gene knockout studies of Krüppel-like factors (KLFs) in mice have demonstrated key roles in organogenesis. EKLF (Klf1) plays an essential role in definitive erythropoiesis and b-globin gene expression, but primitive (yolk sac) erythropoiesis and embryonic globin gene expression are essentially normal. Since expression of embryonic globin genes is dependent upon proximal CACC box elements, additional KLFs must function during the embryonic wave of erythropoiesis. A number of KLFs have been described in zebrafish. One of these, zKLF4, the homologue of neptune, a Xenopus Laevis KLF expressed in the ventral blood island, cement and hatching glands, is an early marker of lateral plate mesoderm (LPM). From 1 somite and prior to expression of scl and flk1, there is a continuous rim of expression of zKLF4 2–3 cells wide within the LPM extending from the postero-lateral boundary of the polster to the tail. Expression in the posterior lateral plate persists as it migrates medially in a zipper-like movement to generate the intermediate cell mass (ICM), the site of embryonic haematopoiesis. zKLF4 is also expressed in a patch of dorsal mesendodermal cells from 70% epiboly which are fated to become the polster. Morpholino knockdown of zKLF4 results in moderate anaemia which resolves as definitive erythropoiesis replaces primitive erythropoiesis at 48–72 hpf. zKLF4 morphants show a down-regulation in the expression of GATA-1 (>100 fold) and embryonic globin (>10 fold) by real time RT-PCR. Both are expressed during the primitive wave of haematopoiesis. In addition there is a dramatic up-regulation (>100 fold) in the level of cmyb expression in zKLF4 morphant compared to control embryos at 72 hpf, which is consistent with expansion of the secondary definitive wave of erythropoiesis, possibly in response to anemia. These results suggest mammalian KLF orthologs of zKLF4, either KLF4 or KLF2, may play roles in primitive erythropoiesis and embryonic globin gene expression in mice or man.

The changing cellular environments of hematopoiesis in human development in utero

The hematopoietic system is indispensable from the earliest stages of development and adapts to the rapidly changing anatomy of the embryo and fetus; this takes place in such different anatomic locations as the yolk sac blood island, hepatic parenchyme, aorta-gonads-mesonephros paravascular mesenchyme, and bone marrow primary logette. We herein summarize our investigation of these serial blood-forming events in the human embryo and fetus. The access to early stages of human development, availability of a large panoply of molecular markers for human blood cell lineages, and recent development of robust assays for the earliest human hematopoietic stem cells have allowed us to gain relatively clear insight into the developmental sequence that underlies the ontogeny of human blood cells. Conversely, the control exerted by these diverse cellular environments on the emergence of human hematopoietic cells remains elusive, as is the case in animal models. We nonetheless present preliminary attempts to decipher the structure and molecular characteristics of the distinct cellular "niches" in which blood cells are produced during human gestation.

Zygotic nucleosome assembly protein–like 1 has a specific, non–cell autonomous role in hematopoiesis

AbstractNucleosome assembly proteins (NAPs) bind core histones, facilitate chromatin remodeling, and can act as transcriptional coactivators. We previously described the isolation of a Xenopus NAP1-like (xNAP1L) cDNA, which encodes a member of this protein family. Its zygotic expression is restricted to neural cells, the outer cells of the ventral blood island (VBIs), and the ectoderm overlying the blood precursors. Here, we report that depletion of zygotic xNAP1L in embryos produces no obvious morphologic phenotype, but ablates α-globin mRNA expression in the VBIs. Transcript levels of the hematopoietic precursor genes SCL and Xaml (Runx-1) are also reduced in the VBIs. SCL expression can be rescued by injection of xNAP1L mRNA into the ectoderm, showing that the effect of xNAP1L can be non–cell autonomous. Fli1 and Hex, genes expressed in hemangioblasts but subsequently endothelial markers, were unaffected, suggesting that xNAP1L is required for the hematopoietic lineage specifically. Our data are consistent with a requirement for xNAP1L upstream of SCL, and injection of SCL mRNA into xNAP1L-depleted embryos rescues α-globin expression. Thus, xNAP1L, which belongs to a family of proteins previously believed to have general roles, has a specific function in hematopoiesis.

Phylogenomic analysis and expression patterns of large Maf genes in Xenopus tropicalis provide new insights into the functional evolution of the gene family in osteichthyans

We have performed an exhaustive characterization of the large Maf family of basic leucine zipper transcription factors in vertebrates using the genome data available, and studied the embryonic expression patterns of the four paralogous genes thus identified in Xenopus tropicalis. Our phylogenetic analysis shows that, in osteichthyans, the large Maf family contains four orthology classes, MafA, MafB, c-Maf and Nrl, which have emerged in vertebrates prior to the split between actinopterygians and sarcopterygians. It leads to the unambiguous assignment of the Xenopus laevis XLmaf gene, previously considered a MafA orthologue, to the Nrl class, the identification of the amphibian MafA and c-Maf orthologues and the identification of the zebrafish Nrl gene. The four X. tropicalis paralogues display partially redundant but nevertheless distinct expression patterns in the somites, developing hindbrain, pronephros, ventral blood island and lens. Comparisons with the data available in the mouse, chick and zebrafish show that these large Maf expression territories are highly conserved among osteichthyans but also highlight a number of differences in the timing of large Maf gene expression, the precise extent of some labelled territories and the combinations of paralogues transcribed in some organs. In particular, the availability of robust phylogenies leads to a reinterpretation of previous expression pattern comparisons, suggesting an important part for function shuffling within the gene family in the developing lens. These data highlight the importance of exhaustive characterizations of gene families for comparative analyses of the genetic mechanisms, which control developmental processes in vertebrates.

An amphibian model to test the effects of xenobiotic chemicals on development of the hematopoietic system

A number of manmade chemicals have deleterious effects on the developing immune system. Very few assay systems are available to study the effects of xenobiotics on hematopoietic stem cells. In rodent models, assays require exposure of pregnant females and analysis of the hematopoietic potential of stem cells from the offspring. These models are less relevant to lower vertebrates such as fish or amphibians where exposure of embryos is direct. To overcome this problem, an amphibian model was developed. Diploid (2N) embryos (16-20 h of age) of the South African clawed frog, Xenopus laevis, were exposed to 10 microg/ml diazinon or 10(-6) M lead acetate for 2 h. After 2 h, the ventral blood island (VBI) was transplanted from a chemically treated or untreated control embryo to an untreated triploid (3N) host embryo. After 55 d, the contribution of the donor VBI-derived stem cells to populations in the blood, thymus, and spleen was assessed by flow cytometry. Diazinon, but not lead acetate, interfered with the ability of transplanted stem cells to contribute to hematopoiesis. Because amphibian embryos are very sensitive indicators of the toxic effects of chemicals, this VBI assay could be employed to test any toxic chemical that is suspected of having a negative effect on development of the hematopoietic system.

Essential role of HGF (hepatocyte growth factor) in blood formation in Xenopus

AbstractIn this study, we investigated the role of hepatocyte growth factor (HGF) in blood formation during Xenopus development. First, we examined the gene expression of HGF and its receptor, c-met, by whole-mount in situ hybridization during development. Strong signals of HGF as well as c-met were detected early in the developing ventral mesoderm, which later gives rise to the ventral blood island. Furthermore, to study the role of HGF, we blocked the HGF signaling pathway in Xenopus embryos by using truncated c-met lacking the tyrosine kinase domain. Injection of truncated c-met mRNA resulted in a marked decrease in the number of circulating blood cells. Similar results were obtained using morpholino antisense HGF oligonucleotides. Moreover, we also analyzed the expression of several early primitive blood markers in the blood island of these embryos. RNA in situ analysis revealed a significant reduction (or absence) of stem cell leukemia (SCL), α-globin, and GATA-1 expression, but not GATA-2 expression. In contrast, no significant difference was observed in the levels of expression of early definitive blood markers, SCL, GATA-2, and GATA-3 in the dorsolateral plate, as analyzed by in situ hybridization. Overall, the present study demonstrated that HGF is necessary for primitive hematopoiesis by regulating the expression of SCL. (Blood. 2004;103:3320-3325)

Primitive erythropoiesis is regulated by Smad-dependent signaling in postgastrulation mesoderm

AbstractThe bone morphogenetic proteins (BMPs) are required for the development of ventral mesoderm, which contributes to the ventral blood island and primitive (yolk sac stage) hematopoiesis. Primitive erythropoiesis is defective when BMP signaling is blocked during gastrulation of Xenopus embryos. This phenotype might be attributed to changes in mesoderm patterning leading indirectly to altered erythropoiesis. We developed an inducible system in order to block BMP signaling in a controlled fashion at later time points in development. For this purpose, an inhibitory Smad, xSmad6, was fused to the estrogen receptor ligand-binding domain. We show that ER-xSmad6 is inactive when expressed in developing embryos, but its activity is induced by estradiol. When induced early in development, ER-xSmad6 causes a dorsalized phenotype, equivalent to overexpression of native xSmad6. When ER-xSmad6 is induced after gastrulation, there is a specific defect in primitive erythropoiesis without any apparent effect on axial patterning. Our results identify an embryonic signal that is Smad-dependent, is required for maintaining expression of GATA-1, and functions within mesoderm and not the overlying ectoderm. Thus, BMP signaling is necessary both during mesoderm patterning and also following early specification events for proper regulation of the primitive erythroid lineage.

Xenopus nucleosome assembly protein becomes tissue-restricted during development and can alter the expression of specific genes

Nucleosome assembly proteins have been identified in all eukaryotic species investigated to date and their suggested roles include histone shuttle, histone acceptor during transcriptional chromatin remodelling and cell cycle regulator. To examine the role of these proteins during early development we have isolated the cDNA encoding Xenopus NAP1L, raised an antibody against recombinant xNAP1L and examined the expression pattern of this mRNA and protein. Expression in adults is predominantly in ovaries. This maternal protein remains a major component of xNAP1L within the embryo until swimming tadpole stages. xNAP1L mRNA is initially throughout the embryo but by gastrula stages it is predominantly in the presumptive ectoderm. Later, mRNA is detected in the neural crest, neural tube, eyes, tailbud and ventral blood islands. In order to test whether xNAP1L has a potential role in gene regulation we overexpressed this protein in animal pole explants and tested the effect on expression of a series of potential target genes. The mRNA encoding the transcription factor GATA-2 was markedly up-regulated by this overexpression. These data support a role for xNAP1L in tissue-restricted gene regulation.

The secreted Frizzled-related protein Sizzled functions as a negative feedback regulator of extreme ventral mesoderm

The prevailing model of dorsal ventral patterning of the amphibian embryo predicts that the prospective mesoderm is regionalized at gastrulation in response to a gradient of signals. This gradient is established by diffusible BMP and Wnt inhibitors secreted dorsally in the Spemann organizer. An interesting question is whether ventrolateral tissue passively reads graded levels of ventralizing signals, or whether local self-organizing regulatory circuits may exist on the ventral side to control cell behavior and differentiation at a distance from the Organizer. We provide evidence that sizzled, a secreted Frizzled-related protein expressed ventrally during and after gastrulation, functions in a negative feedback loop that limits allocation of mesodermal cells to the extreme ventral fate, with direct consequences for morphogenesis and formation of the blood islands. Morpholino-mediated knockdown of Sizzled protein results in expansion of ventral posterior mesoderm and the ventral blood islands, indicating that this negative regulation is required for proper patterning of the ventral mesoderm. The biochemical activity of sizzled is apparently very different from that of other secreted Frizzled-related proteins, and does not involve inhibition of Wnt8. Our data are consistent with the existence of some limited self-organizing properties of the extreme ventral mesoderm.

Adult and embryonic blood and endothelium derive from distinct precursor populations which are differentially programmed by BMP in Xenopus.

Blood and blood vessels develop in close association in vertebrate embryos and loss-of-function mutations suggest common genetic regulation. By the criteria of co-expression of blood and endothelial genes, and lineage tracing of progeny, we locate two distinct populations of progenitors for blood and endothelial cells in developing Xenopus embryos. The first population is located immediately posterior to the cement gland during neurula stages and gives rise to embryonic blood and vitelline veins in the anterior ventral blood island (aVBI), and to the endocardium of the heart. The second population resides in the dorsal lateral plate mesoderm, and contains precursors of adult blood stem cells and the major vessels. Both populations differentiate into endothelial cells in situ but migrate to new locations to differentiate into blood, suggesting that their micro-environments are unsuitable for haematopoietic differentiation. Both require BMP for their formation, even the Spemann organiser-derived aVBI, but individual genes are affected differentially. Thus, in the embryonic population, expression of the blood genes, SCL and GATA2, depend on BMP signalling while expression of the endothelial gene, Xfli1, does not. By contrast, Xfli1 expression in the adult, DLP population does require BMP. These results indicate that both adult and the anterior component of embryonic blood in Xenopus embryos derive from populations of progenitors that also give rise to endothelial cells. However, the two populations give rise to distinct regions of the vasculature and are programmed differentially by BMP.

Common and distinct signals specify the distribution of blood and vascular cell lineages in Xenopus laevis embryos.

In an effort to elucidate the regulatory mechanisms that determine the fate of blood cells and vascular cells in the ventral blood island mesoderm, the embryonic expression of Xtie-2, a Xenopus homolog of the tie-2 receptor tyrosine kinase, was examined. Whole-mount in situ hybridization analysis revealed that Xtie-2 mRNA is expressed at the late tailbud stage within the regions where endothelial precursor cells exist. On the ventral side of embryos, Xtie-2-positive cells are predominantly present just outside the boundary of alpha-globin-positive cells, thus the expression pattern of these two markers seems mutually exclusive. Further experiments revealed that there is a consistent and strong correlation between the induction of Xtie-2 and alpha-globin expression in embryos and explant tissues. First, these two markers displayed overlapping expression in embryos ventralized by the removal of a "dorsal determinant" from the vegetal cytoplasm at the 1-cell stage. Second, expression of both Xtie-2 and alpha-globin were markedly induced in ectodermal explants (animal caps) from embryos co-injected with activin and bone morphogenetic protein (BMP)-4 RNA. Furthermore, both Xtie-2 and alpha-globin messages were strongly positive in dorsal marginal zone explants that had been injected with BMP-4 RNA. In contrast, however, there was a clear distinction in the localization of these two transcripts in embryos dorsalized by LiCl treatment. Distinct localization was also found in the ventral marginal zone (VMZ) explants. Using the VMZ explant system, we demonstrate a role of fibroblast growth factor (FGF) signaling in enhancing the vascular cell marker and reducing the blood cell marker. The present study suggests that the early steps of blood and vascular cell differentiation are regulated by a common BMP-4-dependent signaling; however, distinct factor(s) such as FGF are involved in different distribution of these two cell lineages.

The nodal target gene Xmenf is a component of an FGF-independent pathway of ventral mesoderm induction in Xenopus

The interplay of fibroblast growth factor (FGF) and nodal signaling in the Xenopus gastrula marginal zone specifies distinct populations of presumptive mesodermal cells. Cells in the vegetal marginal zone, making up the presumptive leading edge mesoderm, are exposed to nodal signaling, as evidenced by SMAD2 activation, but do not appear to be exposed to FGF signaling, as evidenced by the lack of MAP kinase (MAPK) activation. However, in the animal marginal zone, activation of both SMAD2 and MAPK occurs. The differential activation of these two signaling pathways in the marginal zone results in the vegetal and animal marginal zones expressing different genes at gastrulation, and subsequently having different fates, with the vegetal marginal zone contributing to ventral mesoderm (e.g. ventral blood island) and the animal marginal zone giving rise to dorsal fates (e.g. notochord and somite). We report here the cloning of a cDNA encoding a novel nuclear protein, Xmenf, that is expressed in the vegetal marginal zone. The expression of Xmenf is induced by nodal signaling and negatively regulated by FGF signaling. Results from animal cap studies indicate that Xmenf plays a role in the pathway of ventral mesoderm induction in the vegetal marginal zone.

Primitive and Definitive Blood Share a Common Origin in Xenopus: A Comparison of Lineage Techniques Used to Construct Fate Maps

Primitive blood constitutes the ventralmost mesoderm in amphibians, and its cleavage-stage origin reveals important clues about the orientation of the dorsal/ventral axis in the embryo. In recent years, investigators employing various lineage-labeling strategies have reported disparate results for the origin of primitive blood in Xenopus [W. D. Tracey, Jr., M. E. Pepling, G. H. Thomsen, and J. P. Gergen (1998). Development 125, 1371–1380; M. C. Lane W. C. Smith (1999). Development 126, 423–434; K. R. Mills, D. Kruep, and M. S. Saha (1999). Dev. Biol. 209, 352–368; A. Ciau-Uitz, M. Walmsley, and R. Patient (2000). Cell 102, 787–796]. These discrepancies must be resolved in order to elucidate early embryonic patterning mechanisms in vivo. We directly compared two of the techniques used to determine the origin of the ventral blood islands and primitive blood, injection of either β-galactosidase mRNA or conjugated dextrans, by coinjecting both tracers simultaneously into individual blastomeres in cleavage-stage embryos. We find that dextrans label progeny efficiently, while β-galactosidase activity is not present in many of the progeny of an injected blastomere, suggesting that mRNA fails to diffuse throughout a blastomere. This result demonstrates that β-galactosidase mRNA fails to meet the criterion for a true lineage label, namely efficient detection of the progeny of a blastomere, and raises questions about interpretations based on mapping the ventral blood islands using Lac Z mRNA as a tracer. We examined the origins of the ventral blood islands and primitive blood from the vegetal region of the marginal zone in regularly cleaving embryos by coinjecting both reporters into C-tier blastomeres. Our results demonstrate that both the ventral blood islands and primitive blood routinely arise from all C-tier blastomeres. Our data, in combination with published mapping results for the dorsal aorta, demonstrate that primitive and definitive blood do not have separate origins at the 32-cell stage in Xenopus. In addition, these results support a proposal to align the dorsal/ventral axis of the mesendoderm with the animal/vegetal axis in pregastrula Xenopus.

XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus

Phagocytic myeloid cells provide the principle line of immune defence during early embryogenesis in lower vertebrates. They may also have important functions during normal embryo morphogenesis, not least through the phagocytic clearance of cell corpses arising from apoptosis. We have identified two cDNAs that provide sensitive molecular markers of embryonic leukocytes in the early Xenopus embryo. These encode a peroxidase (XPOX2) and a Ly-6/uPAR-related protein (XLURP-1). We show that myeloid progenitors can first be detected at an antero-ventral site in early tailbud stage embryos (a region previously termed the anterior ventral blood island) and transiently express the haematopoetic transcription factors SCL and AML. Phagocytes migrate from this site along consistent routes and proliferate, becoming widely distributed throughout the tadpole long before the circulatory system is established. This migration can be followed in living embryos using a 5 kb portion of the XLURP-1 promoter to drive expression of EGFP specifically in the myeloid cells. Interestingly, whilst much of this migration occurs by movement of individual cells between embryonic germ layers, the rostral-most myeloid cells apparently migrate in an anterior direction along the ventral midline within the mesodermal layer itself. The transient presence of such cells as a strip bisecting the cardiac mesoderm immediately prior to heart tube formation suggests that embryonic myeloid cells may play a role in early cardiac morphogenesis.

Identification and expression of Helios, a member of the Ikaros family, in the Mexican axolotl: implications for the embryonic origin of lymphocyte progenitors.

Transcription factors of the Ikaros gene family are critical for the differentiation of T and B lymphocytes from pluripotent hematopoietic stem cells. To study the first steps of lymphopoiesis in the Mexican axolotl, we have cloned the Helios ortholog in this urodele amphibian species. We demonstrated that the axolotl Helios contains a 144-bp deletion at the 5' end of the activation domain. Helios is expressed in both the thymus and spleen but not in the liver of the pre-adult axolotl. During ontogeny, Helios transcripts are detected from neurula stage, before the apparition of the first Ikaros transcripts and the colonization of lymphoid tissues. Interestingly, Helios and Ikaros mRNA are found predominantly in the ventral blood islands of late tail-bud embryos. These results suggest that in contrast to the Xenopus and amniote embryos where two sites of hematopoiesis have been characterized, the ventral blood islands could be the major site of hematopoiesis in the axolotl.

Role of the thrombopoietin (TPO)/Mpl system: c‐Mpl‐like molecule/TPO signaling enhances early hematopoiesis in Xenopus laevis

Multiple organs are induced in the primitive embryonic ectoderm excised from blastula stage Xenopus laevis embryos, under the strict control of mesoderm inducing factors. This in vitro system is useful for exploring the mechanisms of development. In this study, the function of thrombopoietin (TPO)/c-Mpl signaling in the development of hematopoietic cells was investigated. An optimal hematopoietic cell induction system was established to evaluate the influence of growth factors on hematopoiesis. It was found that exogenous TPO enhanced hematopoiesis in explants induced by activin and bone morphogenetic protein (BMP)-4 and increased the number of both erythrocytes and leukocytes in a dose-dependent manner. Addition of anti-c-Mpl antibody completely inhibited the expansion of hematopoietic cells stimulated by TPO, and the antibody specifically recognized blood-like cells. These results demonstrate that TPO acts on hematopoietic progenitors induced in explants and the c-Mpl-like molecule in Xenopus mediates the cellular function of TPO. We also found that forced expression of TPO in embryos promoted hematopoiesis in the ventral blood island and the dorsal-- lateral plate mesoderm. These results suggest that hematopoietic stem and progenitor cells are regulated by TPO/c-Mpl signaling from when they appear in their ontogeny. They also suggest that TPO/c-Mpl signaling play a crucial role in the formation of hematopoietic cells in Xenopus.

The two Xenopus Gbx2 genes exhibit similar, but not identical expression patterns and can affect head formation

Gbx2 homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. Two Gbx2 genes were identified in Xenopus laevis, differing in 13 amino acids, including a change in the homeodomain. Xgbx2a is activated earlier during gastrulation and reaches higher levels of expression while Xgbx2b is expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns.

Neptune, a Krüppel-like transcription factor that participates in primitive erythropoiesis in Xenopus

The specification of the erythroid lineage from hematopoietic stem cells requires the expression and activity of lineage-specific transcription factors. One transcription factor family that has several members involved in hematopoiesis is the Krüppel-like factor (KLF) family [1]. For example, erythroid KLF (EKLF) regulates β-globin expression during erythroid differentiation [2–6]. KLFs share a highly conserved zinc finger-based DNA binding domain (DBD) that mediates binding to CACCC-box and GC-rich sites, both of which are frequently found in the promoters of hematopoietic genes. Here, we identified a novel Xenopus KLF gene, neptune, which is highly expressed in the ventral blood island (VBI), cranial ganglia, and hatching and cement glands. neptune expression is induced in response to components of the BMP-4 signaling pathway in injected animal cap explants. Similar to its family member, EKLF, Neptune can bind CACCC-box and GC-rich DNA elements. We show that Neptune cooperates with the hematopoietic transcription factor XGATA-1 to enhance globin induction in animal cap explants. A fusion protein comprised of Neptune's DBD and the Drosophila engrailed repressor domain suppresses the induction of globin in ventral marginal zones and in animal caps. These studies demonstrate that Neptune is a positive regulator of primitive erythropoiesis in Xenopus.