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Hematopoietic stem cells (HSCs) are commonly used in transplantation therapy to rescue the hematopoietic and immune systems following systemic chemotherapy or irradiation. However, some patients receive inadequate numbers of HSCs and this often results in delayed reconstitution of hematopoiesis and immune function and associated toxicities. We previously demonstrated that a stabilized derivative of prostaglandin (PG) E2 increases vertebrate HSCs both in vivo and in vitro. 16,16-dimethyl PGE2 (dmPGE2) significantly increased HSCs during zebrafish embryogenesis and in the adult marrow following injury. Incubation of murine embryonic stem cells with dmPGE2 during embryoid body differentiation resulted in a dose-dependent increase in hematopoietic colonies, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Finally, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in engrafting cells in a limiting dilution competitive repopulation assay. No negative effects on serial transplantability of HSCs were observed in these animal models. To investigate the therapeutic potential of PGE2 for the amplification of blood stem cells, we exposed human cord blood (hCB) cells to dmPGE2 in vitro and measured the effects on stem and progenitor populations both in vitro and in vivo. Red cell depleted umbilical cord blood specimens, cryopreserved for clinical use, were thawed and divided for parallel processing. Ex vivo treatment of hCB cells for 1 hour with dmPGE2 in dextran/albumin had no negative impact on absolute cell count or the viability and relative distribution of both CD45 and CD34 positive cells compared to vehicle treated control hCB cells. Significantly, hCB treated with dmPGE2 produced enhanced numbers of GM and GEMM colonies in methylcellose CFU-C assays compared to controls. Human CB cells treated ex vivo with dmPGE2 for 1 hour and transplanted at a dose of 20 million live CD45+ cells per recipient were capable of repopulating NOD/SCID mice after sublethal irradiation. In comparative studies at 6 weeks post transplantation, human CD34+ and CD45+ cells could be detected in the marrow (>2%) of dmPGE2 treated (4/8) and control treated (1/6) recipients. Long-term and competitive transplantation experiments to assess the effect of dmPGE2 treatment on functional HSCs are currently in progress. Our data suggests that treatment of human cord blood products with dmPGE2 will be both safe and effective in achieving expansion of hematopoietic stem cells for transplantation in the clinical setting. TE North and W Goessling contributed equally to this work.

Activating mutations in RAS family members are common in myeloproliferative disease (MPD) and acute myeloid leukemia (AML). Because the zebrafish has proven to be an excellent leukemia model and can be used in forward genetic and chemical screens to identify modulators of disease pathways, we developed a transgenic zebrafish model of RAS-induced myeloproliferative disease. Stable transgenic zebrafish lines were created in which the ubiquitous B-actin promoter drives expression of a loxed GFP transgene. Upon CRE-mediated recombination, the loxed GFP cassette is excised and the human kRASG12D transgene is expressed. When mated to hsp70-CRE transgenic zebrafish that express CRE recombinase when the animals are heated to 37C for 1 hour, a cohort of 300 double transgenic embryos was generated. In the heat-shocked group, the medial survival time was 25 days, suggesting that oncogenic RAS expression is lethal in developing zebrafish larvae. Of those animals that survived past this time window, most developed sarcomatous muscle tumors. In contrast, most of the double transgenic zebrafish that did not receive heatshock survived to adulthood. Because the hsp70 promoter is active in development and can be induced by stress, the hsp70-CRE transgenic zebrafish line exhibits low levels of CRE expression even in the absence of heatshock. In the non-heat shocked cohort, 10 of 120 double transgenic zebrafish developed MPD by 66 days of life. Histological examination and fluorescence cytometry analysis revealed an expansion of myeloid cell populations within the kidney, comprising granulocytic and monocytic cells in various stages of differentiation, mimicking myeloproliferative diseases seen in both human and mouse. To specifically induce kRASG12D expression in hematopoietic cells, kidneys were dissected from healthy double transgenic animals; heat shocked ex vivo, and then transplanted into sub-lethally irradiated recipient fish. Upon analysis of transplant animals at three months of age, flow cytometry confirmed that MPD had developed in the recipient fish, however other tumor subtypes were not observed. When kidney marrow from transplant animals having MPD were introduced into sub-lethally irradiated secondary recipients, transplant efficiency was greatly reduced. Taken together these results suggest that, as is seen in MPD in mammals, myeloid cells are not fully transformed in our model. In summary, we show that inducible hkRASG12D expression in zebrafish hematopoietic cells leads to myeloproliferative disease and suggest that this model will allow for the identification of novel pathways responsible for full transformation leading to AML.

The zebrafish mutant gavi was identified in a large-scale chemical mutagenesis screen for embryonic anemia. Two alleles were discovered: gaviHE067 and gaviIT029, both of which have a homozygous recessive pattern of inheritance. The gavi mutants developed severe hypochromic anemia at 48 hours post-fertilization and died by 14 days post-fertilization. To determine the cause of the hypochromic anemia, low resolution genetic mapping was performed which revealed transferrin as a candidate gene. Sequencing of transferrin cDNA from the mutants confirmed that each allele of gavi harbors a mutation that results in aberrant splicing, while quantitative realtime RT-PCR demonstrated virtually absent transferrin expression in the mutants. Our previous work demonstrated that expression of hepcidin, an iron regulatory gene, is upregulated 18 hours following iron dextran injection of the zebrafish embryo. This effect was not impaired by a defect in ferroportin1 (Fraenkelet al., (2005) J Clin Invest115:1532–1541), an iron exporter and ligand for hepcidin. To evaluate the effects of iron loading on transferrin-deficient mutants, gaviIT029 homozygotes at 48 hours post-fertilization were either anesthetized and injected with iron dextran, or anesthetized but not injected. Transferrin expression was assayed 18 hours post-treatment. gaviIT029 homozygote embryos exhibited low levels of hepcidin expression that failed to increase following iron injection. These findings provide the first evidence to support the hypothesis that transferrin is required for the induction of hepcidin expression in response to iron loading in vivo.

HOX genes have emerged as critical effectors of leukemogenesis, but the mechanisms that regulate their expression in leukemia are not well understood. Recent data suggest that the caudal homeobox transcription factors CDX1, 2 and 4, developmental regulators of HOX gene expression, may contribute to HOX gene dysregulation in leukemia. We report here that CDX4 is normally expressed in early hematopoietic progenitors, and is aberrantly expressed in~25% of AML patient samples. Cdx4 regulates Hox gene expression in the adult murine hematopoietic system, and upregulates Hox genes that are implicated in leukemogenesis. Furthermore, bone marrow progenitors that are retrovirally engineered to express Cdx4 serially replate in methylcellulose cultures, grow in liquid culture and generate a partially penetrant, long-latency acute myeloid leukemia (AML) in bone marrow transplant recipients. Co-expression of the Hox co-factor Meis1a accelerates the Cdx4 AML phenotype and renders it fully penetrant. Structure -function analysis demonstrates that leukemic transformation requires intact Cdx4 transactivation and DNA-binding domains, but not the Pbx co-factor interaction motif. Taken together, these data indicate that Cdx4 regulates Hox gene expression in adult hematopoiesis and may serve as an upstream regulator of Hox gene expression in the induction of acute leukemia. Inasmuch as many human leukemias show dysregulated expression of a spectrum of HOX family members, these collective findings also suggest a central role for CDX4 expression in the genesis of acute leukemia.

Hematopoietic stem cells (HSCs) give rise to each of the blood lineages found in the adult vertebrate. Definitive HSCs are derived during embryogenesis in the aorta-gonad-mesonephros (AGM) region and subsequently colonize the niche in fetal and adult hematopoietic organs. To identify regulators of this process, a library of biologically active compounds was screened for effects on zebrafish HSC development. Embryos were exposed to individual chemicals (2498) from the 5-somite stage until 36 hpf, then processed by whole-mount in situ hybridization for the expression of HSC markers cmyb and runx1. Exposure to 47 of the compounds reduced the number of runx1+cmyb+ HSCs, while 35 increased this cell population. Several chemicals isolated in the screen were known to affect the prostaglandin (PG) synthesis pathway. Prostaglandin (PG) precursors, mead acid and linoleic acid, expanded HSCs, while fenbufen and celecoxib, inhibitors of cyclooxygenase (cox) activity, reduced HSC number. An examination of blood and vessel specific molecular markers demonstrated that angiogenesis was not altered significantly in treated embryos, illustrating the specific effect on HSCs. Exposure to exogenous PGE2 modestly increased HSCs, while use of a long-acting version of PGE2, 16,16-dimethylPGE2 (dmPGE2), markedly enhanced HSC number. Incubation of zebrafish embryos with known cox inhibitors confirmed the observation that inhibition of PG synthesis reduces HSCs. Injection of morpholino oligonucleotides designed against cox1, cox2 and PGE synthase inhibited the formation of HSCs in a dose dependent fashion, verifying that signaling through PGE2 is regulating AGM HSC number. Using CD41:GFP;LMO2:DsRed bigenic zebrafish, the modulation of HSCs following exposure to dmPGE2 or indomethacin was documented in vivo by confocal microscopy. Exposure to dmPGE2 following irradiation of adult fish induced HSC gene expression and enhanced hematopoietic recovery, indicating that PGE2 also functions in regulating adult marrow HSC homeostasis. Incubation of murine embryonic stem cells with PGE2 during embryoid body differentiation resulted in a dose-dependent increase of hematopoietic colonies in methylcellulose, while indomethacin exposure inhibited colony formation, demonstrating that the function of PGE2 in HSC regulation is conserved in mammals. Furthermore, ex vivo treatment of murine bone marrow with dmPGE2 resulted in a 2-fold increase in CFU-S number and enhanced the frequency of multi-lineage engrafting HSCs more than 3-fold in a limiting dilution competitive repopulation assay. The identification of the prostaglandin synthesis pathway as a potent regulator of vertebrate hematopoietic stem cell homeostasis has major implications for new treatments of diseases such as anemia and leukemia.

During vertebrate embryonic hematopoiesis, the first blood cells can be identified by expression of the transcription factor genes scl and GATA2, followed by expression of GATA1, a gene required for the erythroid lineage. A high-throughput in situ hybridization screen in zebrafish analyzed the expression pattern of 3700 clones from a hematopoietic cDNA library and discovered 24 genes with expression in the blood. Examination of gene expression in Biklf, GATA1, GATA2, and GATA1/GATA2-deficient animals revealed that most blood genes are dependent upon GATA factors for expression rather than the Krüppel-like transcription factor Biklf. Three novel genes, expressed specifically in erythroid precursors, did not require GATA factors for their expression, demonstrating that some blood genes are regulated in a GATA-independent manner. These three genes were kelch-repeat protein (kelch repeats have been implicated in diverse cellular functions from actin binding to sequestering transcriptions factors), kiaa0650, which contains an SMC-hinge domain, and testhymin, which has no known structural motifs. By using combinations of antisense morpholinos to the known hematopoietic genes biklf , GATA1, GATA2, and scl, we were able to examine the regulation of these novel genes in double and triple knock-down embryos. While expression of kelch-repeat protein was lost in the absence of GATA1 and Biklf, expression of testhymin and kiaa0650 was maintained in GATA1/GATA2/Biklf-deficient embryos, suggesting that these similarly expressed genes are differentially regulated. As with GATA1, kiaa0650 and kelch-repeat protein required Scl for their expression in the early hematopoietic mesoderm while testhymin did not. Furthermore, loss of Scl and GATA2 did not completely ablate testhymin expression, suggesting that this gene is induced by factors upstream or parallel to Scl and GATA2. Taken together, our zebrafish studies establish a regulation of gene expression by a developmental hierarchy of specific transcription factors that act in combination during blood cell maturation.

The differentiation of hematopoietic stem cells (HSCs) into distinct blood lineages is orchestrated by a tight regulation of critical genes during development. To describe the genetic program during the early steps of blood and blood vessel formation in greater detail, we analyzed the expression profiles of zebrafish cloche mutants (clo), in which blood and endothelial cells are completely abrogated, and of isolated hematopoietic and vascular progenitor cell populations from FAC-sorted transgenic lmo2-GFP zebrafish embryos. Consistent with a defect in hemangioblast differentiation, clo mutants at the 5 somite stage were characterized by a prominent downregulation of transcription factors, such as scl and lmo2. As a novel finding, clo mutants at this stage strongly upregulated muscle-related genes, such as myosin, actin, or creatine kinase. FAC-sorted lmo2-GFP cells from 14 somite stage embryos expressed similar hematopoietic transcription factors including scl, lmo2, and gata1. In addition, socs genes, tgfb, fos and jun related genes, and heat shock proteins were also strongly expressed. Comparison with the expression profile of human and mouse fetal liver HSCs revealed a similar regulation between fish and mammals: Expression of 102/168 genes from lmo2-GFP positive cells were also present in mammalian fetal HSCs, indicating an evolutionary highly conserved genetic program of blood formation. In sum, gene expression profiling of zebrafish mutants and of sorted progenitor cells from transgenic zebrafish represent a powerful and versatile tool to define the genetic regulation of vertebrate hematopoietic development.

Acquired and congenital defects in iron metabolism from either deficiency or excess are one of the most common human diseases. Here we present the characterization of the zebrafish frascati mutation, which results in a profound hypochromic anemia and a developmental arrest at the pro-erythroblast stage. Using a positional cloning strategy, we have identified the gene disrupted frascati in mutants as a novel member of the mitochondrial solute transporter family. Members of this family of solute carriers have related tripartite sequence and structure. They function in transporting various metabolites and substrates across the inner mitochondrial membrane. We have verified the identity of the gene in zebrafish by the following criteria: (a) tissue-restricted expression in erythroid progenitors, (b) identification of missense mutations from the frascati five alleles, (c) rescue of anemia by over-expression frascati of cRNA in mutant embryos, and (d) mimicry of anemia using inactivating antisense morpholinos in wildtype embryos. We have also identified the functional ortholog in the mouse which has an analogous tissue and developmental expression pattern. The frascati ortholog in the mouse is highly expressed in fetal liver and adult bone marrow and spleen. The murine frascati transcript and protein are induced during terminal erythroid differentiation in MEL cells treated with either DMSO or HMBA. The over-expression of the mouse frascati cRNA in zebrafish frascati mutant embryos rescued their anemia with equal efficacy as the zebrafish clone. Given the identity of the gene and the requirement for iron in heme biosynthesis in the mitochondria of the developing erythron, we injected exogenous iron-dextran into frascati embryos. The embryos injected with iron-dextran were allowed to develop to 3 days post-fertilization, then stained for hemoglobinized cells with o-dianisidine and genotyped. Using this assay, the anemia caused by frascati the mutation could be partially rescued with exogenous iron supplementation. We therefore propose that the frascati gene functions as the essential transporter for iron importation into the mitochondria for heme biosynthesis and subsequent hemoglobin production in developing erythroid progenitors. Insight into the function of frascati the gene will be directly relevant to our understanding of human disorders of iron deficiency anemia and iron-overload sideroblastic anemia.

The zebrafish mutant kugelig (kgg) is caused by a defect in the caudal-related homeobox gene cdx4 and has a deficit in both ‘primitive’ and ‘definitive’ hematopoietic stem cells. The embryonic anemia in the mutants can be rescued by overexpressing hox genes such as hoxb7a and hoxa9a, but not hoxb8a. This suggests that specific hox genes are required to make mesoderm competent to form blood. To further explore this, we undertook a microarray analysis to identify differentially expressed genes in kgg mutants and wild-type embryos. We found that raldh2, an enzyme required for retinoic acid (RA) production, is overexpressed in kgg mutants during the early stages of blood formation. This data led us to hypothesize that RA may act to suppress blood formation and that the cdx-hox pathway functions to limit RA production, thereby permitting blood formation to occur. To test this, we treated wild-type zebrafish embryos with RA and found that they became severely anemic. Treating kgg embryos with DEAB, a chemical that blocks raldh2 activity, restored hematopoiesis in kgg mutants. Expression of hoxa9a was not rescued in these treated embryos, indicating that RA acts downstream of the hox genes. DEAB also induced an expansion of erythroid cells in wild-type embryos, thus supporting the notion that the levels of RA during development are a critical determinant for blood formation. By performing a time-course rescue experiment, we determined that DEAB is effective when scl + hematopoietic progenitors are first formed from mesoderm, suggesting that RA acts upstream of the blood-inducer scl. In support of this, SCL overexpression rescues GATA-1 expression in embryos treated with RA. We next looked at the effects of DEAB and RA on the formation of mouse hematopoietic progenitors arising from ES cell-derived embryoid bodies (EBs). Addition of DEAB to EBs between days 2 to 3 of development resulted in a 5–8 fold increase in ‘primitive’ erythroid colonies (CFU-Ep), analogous to our results in zebrafish embryos. In contrast, RA treatment caused a general inhibition in the growth of all colony types. Taken together, these results suggest a new model in which suppression of RA by the cdx-hox pathway is necessary for yolk sac hematopoiesis to occur. This model provides an explanation for how hox genes control the spatiotemporal formation of hematopoietic tissue during organogenesis and may shed new light on the pathogenesis of leukemias involving translocations of the cdx , hox, and retinoic acid receptors.

Primitive hematopoiesis in vertebrates initiates in the embryonic yolk sac and yields nucleated erythrocytes and macrophages that later enter circulation. In zebrafish, a group of blood cells contained within the axial vein make up the intermediate cell mass (ICM), the teleost equivalent of the mammalian yolk sac. To identify novel genes involved in hematopoiesis, a high-throughput whole embryo in situ hybridization screen was performed. Examination of the expression pattern of 3700 clones from an adult zebrafish hematopoietic cDNA library discovered 24 genes with expression in the blood during development. Each stage of hematopoiesis was defined by a subset of genes, providing a molecular signature of blood cell maturation, from hematopoietic progenitors to terminally differentiated erythrocytes. By using antisense morpholinos to the transcription factors gata1 and gata2, we were able to dissect the regulation of these 24 genes. Examination of gene expression in Gata1, Gata2, and Gata1/Gata2-deficient animals revealed that most erythroid genes are dependent upon Gata factors for expression. Surprisingly, three novel genes, expressed in hematopoietic progenitors, do not require Gata factors for their expression demonstrating that some erythroid genes are regulated in a Gata-independent manner. During our analysis, we also found persistent ectopic expression of the myeloid transcription factor, PU.1, in the ICM cells and a subsequent expansion of mpo expressing granulocytes and L-plastin expressing macrophages. By utilizing gata1-GFP transgenic zebrafish, we were able to isolate blood cells by flow cytometry and examine their morphology. We discovered that blood cells from the Gata1-deficient animals exhibited features characteristic of myeloid cells when compared to wild-type blood cells. By confocal microscopy, we detected some blood cells in the ICM of Gata1-deficient embryos that co-express globin and PU.1, while blood cells of wild-type embryos never co-express these markers at this stage. These observations demonstrate that in the absence of Gata1 the presumptive erythroid progenitors have transformed into the myeloid lineage, and that a major cell fate alteration has occurred. Ultimately, our studies have molecularly defined blood development by gene expression, and illustrated that Gata1 governs lineage fate decisions of hematopoietic progenitors in the developing embryo.

Iron is required in the mitochondria both to produce heme, which is used for hemoglobin synthesis, and to make iron-sulfur (Fe/S) clusters, which confer electron transfer or catalytic functions to proteins. Cellular iron utilization and Fe/S cluster production are thought to occur independently, yet the processes are coordinated through currently uncharacterized pathways. The shiraz (sir) zebrafish mutant manifests a hypochromic, microcytic anemia. Positional cloning of sir discovered a deletion at the locus that included the zebrafish orthologue to glutaredoxin 5 (grx5), a gene required in yeast for Fe/S cluster assembly. We found that grx5 is highly expressed in the developing blood and fetal liver of both zebrafish and mouse embryos. Antisense-mediated morpholino knockdown of grx5 prevented hemoglobin production, and overexpression of zebrafish, yeast, mouse, or human grx5 RNA in sir embryos completely rescued hemoglobin production, indicating that grx5 is the gene responsible for the sir phenotype. Expression of zebrafish grx5 was found to rescue Fe/S protein production in the yeast Δgrx5 strain, demonstrating that the role of grx5 in Fe/S cluster assembly is conserved among eukaryotes. The surprising finding that mutating a gene necessary for Fe/S cluster assembly caused a lack of hemoglobin synthesis suggested that we had discovered a connection between these pathways. In vertebrates, iron regulatory protein 1 (IRP1) acts as a sensor of intracellular iron levels and controls cellular iron homeostasis via posttranscriptional regulation of iron uptake, storage, and utilization genes. For instance, IRP1 binds to the 5′ iron response element (IRE) in the aminolevulinate synthase 2 (ALAS2) mRNA, blocking translation when cellular iron is low. However, when cellular iron is replete, IRP1 binds a Fe/S cluster and its RNA-binding activity is abolished. We hypothesized that the loss of Fe/S cluster assembly in sir would activate IRP1 and block ALAS2 synthesis, resulting in hypochromia. In support of this model, overexpression of ALAS2 RNA without the 5′ IRE rescued sir hypochromia, while overexpression of ALAS2 including the IRE did not facilitate rescue. Furthermore, antisense morpholino knockdowns of IRP1 caused rescue of hemoglobin synthesis in sir embryos. The combination of these data indicate that hemoglobin production in the differentiating red cell is monitored through Fe-S cluster assembly as a mechanism to gauge iron levels and accordingly direct heme synthesis. This finding illustrates a crucial role for the mitochondrial Fe/S cluster assembly machinery during hemoglobin production, and has broad implications for the role of such genes in human hypochromic anemias.

Vertebrate hematopoiesis can be divided into two embryonic phases: a short primitive wave predominantly generating erythrocytes and a definitive (fetal/adult) wave producing long-term hematopoietic stem cells (HSCs). The definitive wave occurs in the embryonic aorta-gonad-mesonephros (AGM) region through the asymmetric induction of HSCs from the ventral, but not dorsal, aortic endothelial wall. Since Notch signaling is critical for orchestrating a variety of developmental cell fate choices from invertebrates to humans and has been implicated in affecting the differentiation of some hematopoietic lineages, we analyzed whether the Notch pathway regulates definitive HSC induction in vivo. The zebrafish mutant mindbomb harbors a mutation in an essential E3 ligase that ubiquitylates Delta, which in turn allows the Notch intercellular domain to be released and activate downstream target gene transcription. Thus, in the absence of Mindbomb function Notch signaling does not occur. We found that although mindbomb mutants show normal primitive hematopoiesis, definitive c-myb and runx1 HSC expression is lacking. Since embryos injected with synthetic morpholinos designed to inhibit proper splicing of runx1 RNA ( runx morphants) show the same hematopoietic phenotype as mindbomb mutants, we next addressed the epistatic relationship between notch and runx1 using classic gain-of-function and loss-of-function analyses. In runx1 morphants expression of a notch receptor, notch3, and a delta ligand, deltaC, in the developing dorsal aorta was normal. Moreover, injection of runx1 RNA rescued HSCs in the AGM of mindbomb mutants. Together, these results suggest that Runx1 functions downstream of Notch in promoting HSC fate. We next analyzed whether a constitutively activated form of Notch (NICD) is sufficient for HSC specification in the AGM using an inducible binary transgenic system. Zebrafish carrying the heat-shock promoter driving the activator gal4 were mated to animals carrying 6 gal4 -responsive tandem upstream activating sequences (UAS) driving NICD. At the 10 somite-stage the embryos were heat-shocked at 37°C for 1 hour to activate NICD throughout the double transgenic animals. Surprisingly, expression of both HSC markers, c-myb and runx1, were expanded from their normal restricted domain in the ventral endothelium to the entire circumference of the dorsal aorta. Most interestingly, the presence of ectopic c-myb and runx1 transcripts were observed in the developing post-cardinal vein, a vessel that normally does not produce HSCs. These data imply that activation of the Notch pathway generates increased numbers of HSCs in vivo. When runx1 RNA is injected into wild-type embryos a similar expansion of c-myb transcripts is seen throughout the entire dorsal aorta and post-cardinal vein, further indicating that Runx1 functions downstream of Notch in HSC induction. In summary, discovery of the molecular programs essential and sufficient for fetal/adult hematopoietic ontogeny will lead to a further understanding of the physiologic and pathologic processes regulating stem cell homeostasis and translate into more effective therapies for blood disorders.

Patients with type 4 hemochromatosis exhibit iron accumulation in the reticuloendothelial system and a tendency to develop anemia following venesection. These patients are heterozygous for mutations in the intestinal and macrophage iron exporter, ferroportin1 (fpn1), however it is not known whether these mutations cause a gain or loss of function. Of current animal models, only the Tp85c allele of the zebrafish anemia mutant weissherbst (weh) encodes a missense mutation (L167F) in the conserved region of fpn1, between transmembrane helix 3 and 4, where several missense mutations have been identified in individuals with type 4 hemochromatosis. Tp85c homozygote zebrafish die within 10–14 days post-fertilization, however administering iron dextran injections allows them to survive. To explore the effects of fpn1 deficiency on blood development and iron homeostasis in adult zebrafish, we raised Tp85c homozygotes along with control cohorts of injected and uninjected heterozygotes and wild types. Iron dextran injections were discontinued at 4 months of age in all cohorts. Although iron accumulation was observed in all iron-injected fish, only homozygotes exhibited iron accumulation in the intestinal epithelium on Perl’s staining, consistent with a block in enterocyte export. At 6 months of age, none of the cohorts were anemic, however at 12 months of age, only the homozygotes developed hypochromic anemia, characterized by reduced levels of hemoglobin per erythrocyte and impaired late erythroid maturation. Histologic evaluation of the anemic homozygotes revealed increased numbers of iron-loaded macrophages in the liver and in the kidney, the zebrafish bone marrow equivalent. Quantitative realtime rt-PCR, performed on liver and intestinal tissues at 1 year of age, revealed that homozygotes had increased intestinal expression of fpn1 compared to the other cohorts. Hepatic transcript levels of the secreted iron-regulator hepcidin were profoundly decreased in homozygotes, consistent with response to anemia. In contrast, hepatic transcript levels for transferrin, fpn1, and DMT1 were not significantly different among the cohorts. In summary, we provide evidence that Tp85c is a loss of function mutation affecting enterocytes and macrophages. After iron dextran injections were discontinued, Tp85c homozygotes developed anemia, despite increased macrophage iron stores. These studies support the hypothesis that fpn1-deficiency impairs iron cycling and suggest this as a possible mechanism in type 4 hemochromatosis.

In their essay in the April 2004 issue of PLoS Biology, Elizabeth Blackburn and Janet Rowley (2004), two distinguished cellular biologists and members of the President's Council on Bioethics, strongly question the scientific foundation of two reports from the Council (President's Council on Bioethics 2003, 2004). The Council on Bioethics was formed by executive order “to advise the President on bioethical issues that may emerge as a consequence of advances in biomedical science and technology.” An open discussion between ethicists and scientists is critical to the advisory system. The recent administrative dismissal of Dr. Blackburn from the Council is very alarming. By stacking the deck with conservative opinions, and not accurately discussing the scientific issues, the Bioethics Council has become irrelevant to the scientific community and presents a jaundiced view to the public.

The relatively recent availability of human embryonic stem cell (ESC) lines holds tremendous potential for a variety of therapeutic applications. ESCs are self-renewing and pluripotent, capable of generating cells of all tissue types. The ability to coax ESCs in vitro toward lineage-specific

Epo-EpoR signaling is required during definitive hematopoiesis for the proliferation, survival, and differentiation of erythroid progenitors. In addition to its expression in hematopoietic cells, EpoR is also expressed by endothelial cells, neural cells, and cardiac tissue. But the requirement for

Genes expressed in erythroid cells contain binding sites for a cell-specific factor believed to be an important regulator for this haematopoietic lineage. Using high-level transient expression in mammalian cells, we have identified complementary DNA encoding the murine protein. The factor, a new member of the zinc-finger family of DNA-binding proteins, is restricted to erythroid cells at the level of RNA expression and is closely homologous between mouse and man.

Viral infections are frequently associated with haematological disorders (reviewed in ref. 1). Abnormalities including leukopenia, anaemia and thrombocytopenia are commonly observed in patients with the acquired immune deficiency syndrome (AIDS) or the AIDS-related complex (ARC)2–12. The underlying cause of these haematological abnormalities is poorly understood. We report here that bone marrow progenitors isolated from AIDS or ARC patients are responsive to recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF) and recombinant erythropoietin. Antibodies present in the serum of patients infected with the human immunodeficiency virus (HIV), however, could suppress the growth of these progenitors, but not the growth of progenitors from HIV seronegative controls. A component of this immune-mediated suppression appears to be antibodies directed towards the envelope glycoprotein (gp120) of HIV.

An attempt to evaluate the role of the liver in extrarenal erythropoietin production was made by measuring the content of erythropoietin in homogenates and perfusates from hypoxic rat livers. Extracts from livers from nephric or anephric animals rendered both anemic and hypoxic showed no detectable erythropoietin despite the fact that both plasma and kidney extracts contained large amounts of erythropoietin. This lack of measurable erythropoietin in the liver is not caused by degradation of erythropoietin during the extraction procedure because exogenously added rat erythropoietin was recovered to the same extent from livers or kidney homogenates. More likely, however, it is caused by the fact that extrarenal erythropoietin production accounts for only one-fifth of total erythropoietin production and that the liver mass is about six times that of both kidneys. Consequently, the erythropoietin content of 1 g of liver should be about one-thirtieth of that of 1 g of kidney, an amount that is below the limit of detection of the assay. On the other hand, the 2-h in situ perfusates of livers from similarly stimulated animals contained significant amounts of secreted erythropoietin. It is concluded that the liver participates actively in extrarenal erythropoietin production in the adult rat. However, the small amount expected to be present in tissue homogenates cannot be detected with our current bioassay.

SummaryThe zebrafish is a useful model for understanding normal and cancer stem cells, but analysis has been limited to embryogenesis due to the opacity of the adult fish. To address this, we have created a transparent adult zebrafish in which we transplanted either hematopoietic stem/progenitor cells or tumor cells. In a hematopoiesis radiation recovery assay, transplantation of GFP-labeled marrow cells allowed for striking in vivo visual assessment of engraftment from 2 hr–5 weeks posttransplant. Using FACS analysis, both transparent and wild-type fish had equal engraftment, but this could only be visualized in the transparent recipient. In a tumor engraftment model, transplantation of RAS-melanoma cells allowed for visualization of tumor engraftment, proliferation, and distant metastases in as little as 5 days, which is not seen in wild-type recipients until 3 to 4 weeks. This transparent adult zebrafish serves as the ideal combination of both sensitivity and resolution for in vivo stem cell analyses.

Globin gene switching is a complex, highly regulated process allowing expression of distinct globin genes at specific developmental stages. Here, for the first time, we have characterized all of the zebrafish globins based on the completed genomic sequence. Two distinct chromosomal loci, termed major (chromosome 3) and minor (chromosome 12), harbor the globin genes containing α/β pairs in a 5′–3′ to 3′–5′ orientation. Both these loci share synteny with the mammalian α-globin locus. Zebrafish globin expression was assayed during development and demonstrated two globin switches, similar to human development. A conserved regulatory element, the locus control region (LCR), was revealed by analyzing DNase I hypersensitive sites, H3K4 trimethylation marks and GATA1 binding sites. Surprisingly, the position of these sites with relation to the globin genes is evolutionarily conserved, despite a lack of overall sequence conservation. Motifs within the zebrafish LCR include CACCC, GATA, and NFE2 sites, suggesting functional interactions with known transcription factors but not the same LCR architecture. Functional homology to the mammalian α-LCR MCS-R2 region was confirmed by robust and specific reporter expression in erythrocytes of transgenic zebrafish. Our studies provide a comprehensive characterization of the zebrafish globin loci and clarify the regulation of globin switching.

We investigated the potential of mouse embryonic stem (ES) cells to differentiate into hepatocytes in vitro. Differentiating ES cells expressed endodermal-specific genes, such as alpha-fetoprotein, transthyretin, alpha 1-anti-trypsin and albumin, when cultured without additional growth factors and late differential markers of hepatic development, such as tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6P), when cultured in the presence of growth factors critical for late embryonic liver development. Further, induction of TAT and G6P expression was induced regardless of expression of the functional SEK1 gene, which is thought to provide a survival signal for hepatocytes during an early stage of liver morphogenesis. The data indicate that the in vitro ES differentiation system has a potential to generate mature hepatocytes. The system has also been found useful in analyzing the role of growth factors and intracellular signaling molecules in hepatic development.

Despite a long and valuable history, human-animal chimera research has often been questioned. Among the moral issues raised by chimeras is the concept that integration of human cells into anatomical locations such as the brain might endow animals with "human-like" capacities including self-awareness. We present a justification for one type of human-animal chimera experiment: the evaluation of hES cell developmental potency via teratoma formation in immunodeficient mice. We argue that this experiment raises no significant moral concerns and should be the jurisdiction of animal care and use committees and exempt from formal review by the stem cell research oversight process.

In vertebrates, hematopoietic and vascular progenitors develop from ventral mesoderm. The first primitive wave of hematopoiesis yields embryonic red blood cells, whereas progenitor cells of subsequent definitive waves form all hematopoietic cell lineages. In this report we examine the development of hematopoietic and vasculogenic cells in normal zebrafish and characterize defects in cloche and spadetail mutant embryos. The zebrafish homologs of lmo2, c-myb, fli1, flk1, and flt4 have been cloned and characterized in this study. Expression of these genes identifies embryonic regions that contain hematopoietic and vascular progenitor cells. The expression of c-myb also identifies definitive hematopoietic cells in the ventral wall of the dorsal aorta. Analysis of b316 mutant embryos that carry a deletion of the c-myb gene demonstrates that c-myb is not required for primitive erythropoiesis in zebrafish even though it is expressed in these cells. Both cloche and spadetail mutant embryos have defects in primitive hematopoiesis and definitive hematopoiesis. The cloche mutants also have significant decreases in vascular gene expression, whereas spadetail mutants expressed normal levels of these genes. These studies demonstrate that the molecular mechanisms that regulate hematopoiesis and vasculogenesis have been conserved throughout vertebrate evolution and the clo and spt genes are key regulators of these programs.

Summary Recent genome-wide studies have demonstrated that pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1γ mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1γ -deficient animals. Biochemical analysis established physical interactions among TIF1γ, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. Chromatin immunoprecipitation assays in human CD34 + cells supported a TIF1γ-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.

Chromosomal translocations disrupting the Mixed lineage leukemia ( Mll ) gene result in leukemia, with aberrant expression of some native Mll target genes (reviewed in [1]). The Mll gene encodes a Trithorax-group chromatin regulator that is essential for the development of hematopoietic stem cells (HSCs) during embryogenesis [2]. Like Trithorax, MLL positively regulates clustered homeodomain or Hox genes, yet the role of Hox genes collectively in the development of the mammalian hematopoietic system has been difficult to ascertain because of redundancy among Hox paralogs. Here, we show that in the absence of MLL, early hematopoietic progenitors develop despite reduced expression of HoxA , HoxB , and HoxC genes. However, these progenitors exhibit a marked reduction in their ability to generate hematopoietic colonies, a subsequent process requiring cell division and differentiation. Reactivation of a subset of Hox genes or, remarkably, reexpression of a single Hox gene in Mll -deficient progenitors rescued hematopoietic-colony frequency and growth. In contrast, expression of other MLL target genes such as Pitx2 or expression of anti-apoptotic BCL-2 failed to rescue hematopoietic-colony frequency. Furthermore, our results highlight a shared function of Hox proteins at this point in the development of the hematopoietic system.

SummaryLive imaging of the interactions between oncogene-transformed cells and leukocytes in zebrafish reveals that PGE2 promotes the survival and proliferation of cancer cells. Non-steroid anti-inflammatory drugs, like aspirin, are the effective inhibitors of PGE2 production and could be used with other anti-tumor agents in the treatment of cancer.

Melanoma is the most lethal form of skin cancer, and the incidence and mortality rates are rapidly rising. Epidemiologically, high numbers of nevi (moles) are associated with higher risk of melanoma [1]. The majority of melanomas exhibit activating mutations in the serine/threonine kinase BRAF [2–4]. BRAF mutations may be critical for the initiation of melanoma [5]; however, the direct role of BRAF in nevi and melanoma has not been tested in an animal model. To directly test the role of activated BRAF in nevus and melanoma development, we have generated transgenic zebrafish expressing the most common BRAF mutant form (V600E) under the control of the melanocyte mitfa promoter. Expression of mutant, but not wild-type, BRAF led to dramatic patches of ectopic melanocytes, which we have termed fish (f)-nevi. Remarkably, in p53-deficient fish, activated BRAF induced formation of melanocyte lesions that rapidly developed into invasive melanomas, which resembled human melanomas and could be serially transplanted. These data provide direct evidence that BRAF activation is sufficient for f-nevus formation, that BRAF activation is among the primary events in melanoma development, and that the p53 and BRAF pathways interact genetically to produce melanoma.

SummaryHematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization.PaperFlick

The regulation of hematopoiesis is generally evolutionarily conserved from zebrafish to mammals, including hematopoietic stem cell formation and blood cell lineage differentiation. In zebrafish, primitive granulocytes originate at two distinct regions, the anterior lateral plate mesoderm (A-LPM) and the intermediate cell mass (ICM). Few studies in the zebrafish have examined genes specifically required for the granulocytic lineage. In this study, we identified the responsible gene for a zebrafish mutant that has relatively normal hematopoiesis, except decreased expression of the granulocyte-specific gene mpx. Positional cloning revealed that phospholipase C gamma-1 (plcg1) was mutated. Deficiency of plcg1 function specifically affected development of granulocytes, especially the maturation process. These results suggested that plcg1 functioned specifically in zebrafish ICM granulopoiesis for the first time. Our studies suggest that specific pathways regulate the differentiation of the hematopoietic lineages.

SummaryFerroportin (SLC40A1) is an iron transporter postulated to play roles in intestinal iron absorption and cellular iron release. Hepcidin, a regulatory peptide, binds to ferroportin and causes it to be internalized and degraded. If ferroportin is the major cellular iron exporter, ineffective hepcidin function could explain manifestations of human hemochromatosis disorders. To investigate this, we inactivated the murine ferroportin (Fpn) gene globally and selectively. Embryonic lethality of Fpnnull/null animals indicated that ferroportin is essential early in development. Rescue of embryonic lethality through selective inactivation of ferroportin in the embryo proper suggested that ferroportin has an important function in the extraembryonic visceral endoderm. Ferroportin-deficient animals accumulated iron in enterocytes, macrophages, and hepatocytes, consistent with a key role for ferroportin in those cell types. Intestine-specific inactivation of ferroportin confirmed that it is critical for intestinal iron absorption. These observations define the major sites of ferroportin activity and give insight into hemochromatosis.

A variety of haematologic abnormalities are associated with infection by HIV, the human retrovirus that is the primary aetiologic agent of the acquired immunodeficiency syndrome (AIDS). We have reviewed the haematologic findings in well-characterized cohorts of patients with AIDS, AIDS-related complex (ARC) and asymptomatic homosexual men at risk for this retrovirus. Anaemia, granulocytopenia and thrombocytopenia were found in increasing prevalence according to the severity of clinical disease associated with retroviral infection. Bone marrow aspirations and biopsies revealed frequent hypercellularity, dysplasia, plasmacytosis and lymphoid infiltrates. These marrow morphologic findings were strongly associated with anaemia and granulocytopenia. Review of transfusion records of patients with HIV antibodies revealed a 21% prevalence of a positive direct antiglobulin test. The pathophysiology of the observed haematologic abnormalities may involve direct retroviral infection of bone marrow progenitors, abnormal regulation of haematopoiesis and/or autoimmune phenomena.

Of 45 individuals seropositive for human T cell lymphotropic virus type III/lymphadenopathy-associated virus, 45 were found to have detectable salivary antibodies to viral antigens by a radioimmunoprecipitation assay. The results also showed that a Western blot assay for salivary antibodies may be possible. The feasibility of a diagnostic test for human T cell lymphotropic virus type III/lymphadenopathy-associated virus not requiring venipuncture is discussed.

Hemophilus parainfluenzae, a common upper respiratory tract pathogen, has been reported to cause pharyngitis, epiglottitis, otitis media, conjunctivitis, and pneumonia. Rarely H. parainfluenzae infects the urinary tract, and is believed not to have been previously reported as a cause of prostatitis. A case of H. parainfluenzae in a young homosexual man infected with HTLV-III and chronic lymphadenopathy is described. Common clinical syndromes such as prostatitis may be associated with unusual pathogens in persons immunodeficient due to HTLV-III infection.

Whole saliva samples collected from available people at risk in Boston for infection with human T-lymphotropic virus type III (HTLV-III/LAV), from late 1984 through early 1985, were analyzed for the presence of antibodies to viral proteins. Fourteen of 20 (70%) acquired immunodeficiency syndrome (AIDS) patients and 14 of 15 (93%) AIDS- related complex (ARC) patients had salivary antibodies that reacted with the virus-encoded glycoproteins gp160 and gp120 of HTLV-III infected cells. All of the AIDS and ARC patients had serum antibodies to the same antigens. Of 20 sex partners of AIDS/ARC patients, nine (45%) showed anti-HTLV-III antibodies, and four of 18 (22%) healthy homosexual males also were positive for such antibodies. Serum and salivary antibody status were the same in these groups. A minority of those patients positive for salivary antibodies to env gene-encoded gp160 and gp120 also had salivary antibodies to gag gene-encoded proteins of 55,000, 24,000, and/or 17,000 daltons. Immunoglobulin A (IgA) class antibodies comprised the majority of the salivary antibody response. The spectrum of HTLV-III proteins detected by the salivary and serum antibodies was similar. The possibility that secretory IgA from the gut-associated lymphoid system may play a role to restrict salivary transmission of HTLV-III should be considered.

Spectrins are key cytoskeleton proteins with roles in membrane integrity, cell morphology, organelle transport and cell polarity of varied cell types during development. Defects in erythroid spectrins in humans result in congenital hemolytic anemias with altered red cell morphology. Although well characterized in mammals and invertebrates, analysis of the structure and function of non-mammalian vertebrate spectrins has been lacking. The zebrafish riesling (ris) suffers from profound anemia, where the developing red cells fail to assume terminally differentiated erythroid morphology. Using comparative genomics, erythroid β-spectrin (sptb) was identified as the gene mutated in ris. Zebrafish Sptb shares 62.3% overall identity with the human ortholog and phylogenetic comparisons suggest intragenic duplication and divergence during evolution. Unlike the human and murine orthologs, the pleckstrin homology domain of zebrafish Sptb is not removed in red cells by alternative splicing. In addition, apoptosis and abnormal microtubule marginal band aggregation contribute to hemolysis of mutant erythrocytes, which are features not present in mammalian red cells with sptb defects. This study presents the first genetic characterization of a non-mammalian vertebrate sptb and demonstrates novel features of red cell hemolysis in non-mammalian red cells. Further, we propose that the distinct mammalian erythroid morphology may have evolved from specific modifications of Sptb structure and function.

Genes expressed in erythroid cells contain binding sites for a cell-specific nuclear factor, GF-1 (NF-E1, Eryf 1), believed to be an important transcriptional regulator. Previously we characterized murine GF-1 as a 413-amino acid polypeptide containing two cysteine-cysteine regions reminiscent of zinc-finger DNA-binding domains. By cross-hybridization to the finger domain of murine GF-1 we have isolated cDNA encoding the human homolog. Peptide sequencing of purified human GF-1 confirmed the authenticity of the human cDNA. The predicted primary sequence of human GF-1 is highly similar to that of murine GF-1, particularly in the DNA-binding region. Although the DNA-binding domains of human, murine, and chicken proteins are remarkably conserved, the mammalian polypeptides are strikingly divergent from the avian counterpart in other regions, most likely those responsible for transcriptional activation. By hybridization to panels of human-rodent DNAs we have assigned the human GF-1 locus to Xp21-11. The localization of the gene to the X chromosome has important implications for hereditary persistence of fetal hemoglobin syndromes unlinked to the beta-globin cluster and for genetic experiments designed to test the role of the factor in erythroid cell gene expression.

Abstract 860 During bone marrow transplantation, hematopoietic stem/progenitor cells (HSPCs) are exposed to various stress signals, and undergo homing, rapid proliferation and differentiation in order to achieve engraftment. To explore how fate decisions are made under such stress conditions, we developed a novel imaging-based competitive marrow transplantation in zebrafish. The feasibility of handling hundreds of zebrafish for transplantation per day allowed us to screen a library of 480 small molecules with known bioactivity, aimed at identifying new drugs and pathways regulating HSPC engraftment. Two structurally related eicosanoids, 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, were able to enhance GFP+ marrow engraftment compared to DsRed2+ engraftment in zebrafish. This remarkable effect of EETs on adult marrow prompted us to study the effect of EETs in embryonic hematopoiesis. Treating zebrafish embryos with 11,12-EET during definitive hematopoiesis increased the HSPC marker Runx1 expression in the AGM (Aorta-Gonad-Mesonephros), resulting in a significant increase of HSPC in the next hematopoietic site, caudal hematopoietic tissue, the equivalent of fetal liver/placenta in mammals. The same treatment condition also induced ectopic Runx1 expression in the tail mesenchyme, a non-hematopoietic tissue. Microarray analysis on EET-treated zebrafish embryos revealed an upregulation of genes involved in stress response, especially Activator Protein 1 (AP-1) family members. Genetic knockdown experiments confirmed AP-1 members, especially JunB and its binding partners, cFos and Fosl2, are required for Runx1 induction. Motif analysis also predicted several conserved AP-1 binding sites in the Runx1 enhancer regions. To understand how EETs induced AP-1 expression, a suppressor screen was performed in zebrafish embryos. The screen revealed that activation of both PI3K/Akt and Stat3 are required for induced AP-1 expression, and therefore Runx1 upregulation. Similarly, ex vivo treatment of mouse whole bone marrow with 11,12-EET resulted in a 2-fold increase of long-term repopulating units. Microarray data had previously shown that Cyp2j6, one of the cytochrome P450 enzymes involved in EET biosynthesis from arachidonic acid, is enriched in quiescent mouse long-term HSCs. To further increase the EET levels in HSPCs, human CYP2C8 enzyme was over-expressed in transgenic mice using the Tie2 promoter. These transgenic mice have a 4-fold increase of long-term multi-lineage repopulating unit compared to their wild-type siblings. In purified mouse HSPCs, EETs directly and cell-autonomously activate PI3K/AKT pathway. Co-treatment of mouse bone marrow with EET and a PI3K inhibitor, LY294,002, completely blocked EET-induced enhancement of mouse bone marrow engraftment. In conclusion, we performed the first competitive marrow transplantation-based chemical screen, leading to the discovery of arachidonic acid-cytochrome P450-EETs as a novel modulator of HSC cell fate decision. PI3K/Akt and Stat3 pathways activated by EETs are required for adult HSPC engraftment and/or embryonic HSC specification, partially through transcriptional regulation of AP-1. We also demonstrated the requirement of AP-1 family members for Runx1 expression during embryonic development. This discovery may have clinical application in marrow or cord blood transplantation. Disclosures: Daley: iPierian, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Epizyme, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Verastem, Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Solasia, KK: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; MPM Capital, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees. Zon:Fate Therapeutics: Founder; Stemgent: Consultancy.