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Genetics of erythropoiesis: Induced mutations in mice and zebrafish
Abstract
Production of red blood cells (erythropoiesis) in the vertebrate embryo is critical to its survival and subsequent development. As red cells are the first blood cells to appear in embryogenesis, their origin reflects commitment of mesoderm to an hematopoietic fate and provides an avenue by which to examine the development of the hematopoietic system, including the hematopoietic stem cell (HSC). We discuss the genetics of erythropoiesis as studied in two systems: the mouse and zebrafish (Danio rerio). In the mouse, targeted disruption has established several genes as essential at different stages of hematopoiesis or erythroid precursor cell maturation. In the zebrafish, numerous mutants displaying a wide range of phenotypes have been isolated, although the affected genes are unknown. In comparing mouse knockout and zebrafish mutant phenotypes, we propose a pathway for erythropoiesis that emphasizes the apparent similarity of the mutants and the complementary nature of investigation in the two species. We speculate that further genetic studies in mouse and zebrafish will identify the majority of essential genes and define a regulatory network for hematopoiesis in vertebrates.
... Genes discovered by analysis of chromosomal translocations and deletions associated with leukemia have been associated with essential proliferation and/ or differentiation decisions in haematopoietic cells (Rabbitts, 1994). Loss of function and gain of function experiments have identified a number o f genes critical for eiythropoiesis (Orkin and Zon, et al, 1997). Indeed, some of these genes, such as SCL, are involved at early stages in haematopoietic development (Yamaguchi, et al., 1993;Gering, et al, 1998). ...
... Indeed, some of these genes, such as SCL, are involved at early stages in haematopoietic development (Yamaguchi, et al., 1993;Gering, et al, 1998). A preliminary hierarchy of transcriptional regulators with distinct and overlapping patterns of expression in erythroid progenitors and precursors has been fashioned from these studies (Orkin and Zon, 1997). ...
The gene that encodes the Lymphoblastic Leukemia protein 1 (LYL-1) (Cleary, et al, 1988) was identified originally through the characterisation of a tumor specific t(7;19) chromosomal translocation involving the T-cell receptor (TCR) C[beta] gene. The LYL-1 protein is a member of the class II basic helix-loop-helix (bHLH) family of transcriptional factors (Mellentin, et al, 1989; Massari and Murre, 2000). LYL-1 forms heterodimers, both in-vitro and in-vivo, with class I bHLH proteins E2A, and HEB (Miyamoto, et al, 1996), and has been shown to interact with motifs present in the NFκB1 precursor p105 (Ferrier, et al, 1999). LYL-1 mRNA is expressed primarily within the haematopoietic system (Kuo, et al, 1991) and displays a pattern of expression that overlaps considerably with stem cell leukemia protein (SCL) (Visvader, et al, 1991). Both SCL and LYL-1 mRNA are expressed in erythroid and myeloid cell lineages as well as in megakaryocytes, but not in normal T-cells (Mellentin, et al, 1989; Mouthon, et al, 1993). LYL-1, unlike SCL, is expressed in cell lines established from leukemias of B lymphocyte origin (Visvader, et al, 1991; Miyamoto, et al, 1996). The amino acid sequence of the LYL-1 protein shares 78% overall identity with it's human counterpart (Visvader, et al, 1991; Kuo, et al, 1991) and possess an identical bHLH domain except for a conservative amino acid substitution in the loop region. Although the role of LYL-1 protein in the oncogenic transformation of T-cells has not been defined, as a consequence of the translocation it's de-regulated expression may alter the precise balance of transcriptional regulators, and thereby precipitate leukaemogenic events in T-cell. The almost identical E-box DNA-binding site for SCL and LYL-1 bHLH heterodimers (Miyamoto, et al., 1996), in conjunction with the critical function SCL haematopoiesis (Begley and Green, 1999), suggests a role for LYL-1 within the haematopoietic cells. The function of LYL-1 in haematopoiesis was examined by disruption of the LYL-1 gene by homologous recombination in ES cells. A lacZ/ neomycin gene cassette was cloned in-frame into the fourth exon, replacing a 0.7kb Hpal fragment encoding the bHLH, as well as the entire 3'- end of die LYL-1 gene. The absence of a functional LYL-1 protein was associated with increased numbers of c-Kit-positive cells, early (BFU-e) and late (CFU-e) erythroid progenitors (approximately 3-5 times) in the spleen. These progenitors generated haemoglobinised erythroblasts in vitro. More BFU-e in the LYL-1−/− spleen responded to Epo only, and in the presence of Epo and SCF some of these BFU-e produced colonies that comprised a large number of erythroid cells. In vivo, splenic erythroid progenitors and precursors expressed SCL mRNA, GATA-1, TER-119, and haemoglobin, and were distributed throughout the red pulp. LYL-1−/− spleen comprised 5 to 10-fold more erythroblasts than normal, which were arranged into clusters that often contained megakaryocytes. There was a 2-fold increase in the number of CFU- GEMM in the LYL-1−/− spleen, but no change in CFU-G. The bone marrow of LYL-1−/− mice contained 1.4-fold more CFU-e compared to LYL-1+/+ controls, but there was no change in BFU-e number. The erythroblasts in the LYL-1−/− spleen were not apoptotic. The loss of a functional LYL-1 protein was further associated with a reduction in the number of erythrocytes, and the amount of stored iron, in the spleen, but no change in the number of erythrocytes in the peripheral circulation, or in the amount of stored iron in the bone marrow. The results presented suggest that LYL-1 has a role in events associated with mobilisation of haematopoietic progenitors from the bone marrow. Expression of a non-functional LYL-1 protein in LYL-1−/− mice induced mobilisation of erythroid progenitors, followed by their expansion in the spleen.
... The -globin gene is highly transcribed in the fetal liver after E12 and is silenced in the fetal brain. 31 There is a marked enrichment of dimethyl H4R3 at the  maj -promoter in fetal liver cells ( Figure 1B), but not in transcriptionally silent fetal brain cells ( Figure 1C). A small but significant enrichment of dimethyl H4R3 is also found between HS2 and HS3 of the LCR in the fetal liver ( Figure 1B). ...
... Furthermore, H4R3 methylation is not detected at the ⑀ y and h1 genes ( Figure 1B), which are transcribed only at the yolk sac stage of erythropoiesis before E12 and are silent in the fetal liver. 31 In addition, there is no enrichment of dimethyl H4R3 at the promoter regions of the housekeeping gene GAPDH and the silenced MyoD gene ( Figure 1B), suggesting that dimethyl H4R3 is important for activation of -globin gene. ...
Histone modifications play an important role in the process of transcription. However, in contrast to lysine methylation, the role of arginine methylation in chromatin structure and transcription has been underexplored. The globin genes are regulated by a highly organized chromatin structure that juxtaposes the locus control region (LCR) with downstream globin genes. We report here that the targeted recruitment of asymmetric dimethyl H4R3 catalyzed by PRMT1 (protein arginine methyltransferase 1) facilitates histone H3 acetylation on Lys9/Lys14. Dimethyl H4R3 provides a binding surface for P300/CBP-associated factor (PCAF) and directly enhances histone H3 acetylation in vitro. We show that these active modifications are essential for efficient interactions between the LCR and the beta(maj)-promoter as well as transcription of the beta-globin gene. Furthermore, knockdown (KD) of PRMT1 by RNA interference in erythroid progenitor cells prevents histone acetylation, enhancer and promoter interaction, and recruitment of transcription complexes to the active beta-globin promoter. Reintroducing rat PRMT1 into the PRMT1 KD MEL cells rescues PRMT1 binding, beta-globin transcription, and erythroid differentiation. Taken together, our data suggest that PRMT1-mediated dimethyl H4R3 facilitates histone acetylation and enhancer/promoter communications, which lead to the efficient recruitment of transcription preinitiation complexes to active promoters.
... In addition, bacterial infections can be analyzed in real-time in zebrafish embryos [5]. This fish is a unique and important animal model in which the power of mutagenesis is applied to the study of vertebrate development [6,7]. Zebrafish genome mutagenesis has provided important insights into genes related to cardiac, vascular, and erythrocyte development, including models of disease such as congenital sideroblastic anemia and hepatoerythropoietic porphyria6789. ...
... This fish is a unique and important animal model in which the power of mutagenesis is applied to the study of vertebrate development [6,7]. Zebrafish genome mutagenesis has provided important insights into genes related to cardiac, vascular, and erythrocyte development, including models of disease such as congenital sideroblastic anemia and hepatoerythropoietic porphyria6789. Given the development of appropriate screening assays, the power of the zebrafish model can be harnessed for the study of other vertebrate functions such as hemostasis, thrombosis and sepsis10111213. ...
We have studied the normal and sepsis effected zebrafish eyeball with micro-magnetic resonance imaging (micro-MRI). T2 weighted image was studied and pixel-wise T2 maps were co registered among the normal and sepsis effected eye. From the micro-image the sepsis effect in the eye has been demonstrated. The pixel-wise brightness distribution is not so scattered in the normal eyeball image, whereas it is very scattered in case of sepsis affected eyeball image. Also the T2 mapping on the eye has given valuable information that would be a potential tool for the study of diseased organ in the micro level. From T2 mapping, it has shown that the T2 in normal eyeball have low values in comparison to the sepsis affected eyeball.
... The zebrafish is a unique and important animal model in which the power of saturation mutagenesis is applied to the study of vertebrate development (4). This approach allows the identification of genes in an "unbiased " manner, as opposed to the gene by gene approach employed by knockout studies in the mouse (5). Theoretically, saturation mutagenesis can identify most of the genes that contribute significantly to a given pathway, which may result in identification of novel factors or previously unrecognized functions for known genes. ...
... Theoretically, saturation mutagenesis can identify most of the genes that contribute significantly to a given pathway, which may result in identification of novel factors or previously unrecognized functions for known genes. Mutagenesis of the zebrafish genome has yielded important insights into genes involved in cardiac, vascular, and erythrocyte development, including models of disease such as congenital sideroblastic anemia and hepatoerythropoietic porphyria (4)(5)(6)(7). Given the development of appropriate screening assays, the power of the zebrafish model can be harnessed for the study of other vertebrate functions such as hemostasis (8)(9)(10). ...
The zebrafish (Danio rerio) is a unique animal model in which saturation mutagenesis has been used to identify genes involved in vertebrate development. The relevance of the zebrafish as a genetic model for hemostasis depends, in large part, on the degree of similarity between the zebrafish and mammalian systems. The diminutive size of the zebrafish poses technical problems for analysis of coagulation. This study describes methods to obtain citrated whole blood and plasma from the zebrafish, analyze in vitro coagulation in small plasma volumes, obtain uniform dosing of zebrafish with oral anticoagulants, and demonstrate specific factor activities via chromogenic assays. Analysis of the zebrafish system demonstrates the presence of both the intrinsic and extrinsic pathways of coagulation, evidence for prothrombin, factor X, protein C, antithrombin, and heparin cofactor II activity, and a requirement for vitamin K dependent γ-carboxylation of zebrafish hemostatic proteins. Induction of a morphologically recognizable bleeding phenotype by warfarin treatment is also demonstrated. Characterization of zebrafish coagulation provides evidence that major hemostatic pathways are conserved between zebrafish and man. These similarities indicate that the zebrafish is a relevant genetic model for identification of novel genes involved in hemostasis and thrombosis.
... There are various advantages in employing zebrafish models for studying hemostasis and have been described in the previous sections. Zebrafish has emerged as a model organism to (1) elucidate the molecular pathways involved in the generation and action of various gene products, (2) validate the genetic causes identified in human population, (3) develop genetic and chemical screens for identification of novel mediators responsible for hemostasis and thrombosis, and (4) identify genes via an unbiased approach rather than gene-by-gene approach as in mice (Orkin and Zon 1997;Santoriello and Zon 2012;Haffter et al. 1996). Zebrafish models for studying hematopoiesis are well established, and features of these model systems like high fecundity, transparent embryos, and easy accessibility of hematopoietic and circulatory system offer better features to study novel features of hemostasis in zebrafish. ...
Tumor angiogenesis is the most crucial step in the progression of all types of cancers. Preexisted blood vessel vascularizes into new one through sprouting or intussusceptive (splitting) mechanism. This process would facilitate the growth in the size of tumors regulated by VEGF (vascular endothelial growth factor), leading to metastasis, which ultimately increases the severity of cancer. So, it is very important to suppress tumor angiogenesis before the situation gets worse in a cancer patient. A wide variety of in vitro and in vivo models have been used to study the process of tumor angiogenesis and metastasis of cancer. It has helped us to discover new drugs and to find novel therapies for cancer, including anti-angiogenic therapy. Mainly angiogenesis is traditionally modeled in rodents and chick embryo, but of late zebrafish is emerging as the preferred model due its several advantages over the other animals. Zebrafish (Danio rerio) serves as the ideal model to study the various cancers, since it is possible to induce tumor growth or suppression easily, when compared to the other animal models. Also, tumor xenograft model has been studied in zebrafish extensively using many human cancer cell lines. So, in this chapter, we have reviewed some literatures that appreciate zebrafish model to study tumor angiogenesis.KeywordsZebrafishAngiogenesisVEGFTumorXenograftAnti-angiogenic therapy
... Zachodzi tam erytropoeza [23, 61]. Po około 24 godzinach od zapłodnienia erytroblasty wędrują do pęcherzyka żółtkowego, gdzie zaczynają krążyć i przenikają do krwi obwodowej [6,46,48]. Dopiero po 4 dniach od zapłodnienia przekształcą się w embrionalne erytrocyty zawierające hemoglobinę[6]. Wyjątek stanową antarktyczne ryby Trematomus bernacchii (Nototheniidae) i Chionodraco hamatus (Channicthyidae), których erytrocyty wcale nie zawierają hemoglobiny[43,50]. ...
Haematopoiesis is a complex process in which haematopoietic stem cells, the most immature elements of the haematopoietic hierarchy, proliferate and differentiate into various classes of haematopoietic progenitor cells. These progenitor cells have been shown to be able to differentiate into mature blood cells: erythrocytes, lymphocytes, thrombocytes, granulocytes, and monocytes. The pronephros, or head kidney, is a basic organ forming the blood elements, and is also a reservoir of blood cells. Basic haematopoietic structures and mechanisms in fish are similar to those functioning in other vertebrates, and all haematopoietic cell types are very similar to those of mammals.
... From an evolutionary perspective, mammalian red blood cells (RBCs) are enucleated (Orkin and Zon, 1997) to increase hemoglobin levels (Ji et al., 2011). Because the committed production of the erythroid lineage via definitive erythropoiesis uniquely includes nuclear extrusion in mammals, the red cell population in the peripheral circulation of, say, adult rodents or humans consists entirely of nucleus-free erythrocytes. ...
Nucleated circulating red blood cells (RBCs) of developing zebrafish, chick and mouse embryos can actively proliferate. While marrow- or organ-mediated erythropoiesis has been widely studied, transforming in vivo processes of circulating RBCs are under little scrutiny. We employed confocal, stereo- and electron microscopy to document the maturation of intravascular RBCs. In zebrafish embryos (32-72 hours post fertilization), RBC splitting in the caudal vein plexus follows a four-step program: (i) Nuclear division with continued cytoplasmic connection between somata. (ii) Dumbbell-shaped RBCs tangle at transluminal vascular pillars. (iii) Elongation, and (iv) Disruption of soma-to-soma connection. Dividing RBCs of chick embryos, however, retain the nucleus in one of their somata. Here, RBC splitting acts to pinch-off portions of cytoplasm, organelles and ribosomes. Dumbbell-shaped primitive RBCs re-appeared as circulation constituents in mouse embryos. The splitting of circulating RBCs, thus, represents a biologically relevant mechanism of RBC division and maturation during early vertebrate ontogeny.
... At E7.5, primitive erythroblasts, macrophages, and a few megakaryocytes are found in blood islands [20][21][22] . Erythrocytes found in the yolk sac are commonly nucleated, larger in size than erythrocytes in postnatal blood, and express different hemoglobins than postnatal erythrocytes 23,24 . As in the formation of hematopoietic cells from the mesoderm in the embryo proper, BMP signaling in the extraembryonic mesoderm is critical for the specification of yolk sac mesoderm to hematopoietic cells 25 . ...
... Erythropoiesis occurs in two distinct phases in zebrafish, primitive and definitive, which differ both temporally and spatially (see Orkin and Zon, 1997). In addition to the apparent lack of definitive erythrocytes, there was evidence of a decreased number of primitive erythrocytes at 36 hpf in retene-exposed zebrafish embryos {Supplemental video: bloodflow_36hpf.mov}. ...
... On the other hand, hematopoietic stem cells (HSCs) develop from the hemogenic endothelium, which is originally derived from endothelial cells (ECs) of the embryonic vasculature (3). In addition, hypoxia also induces erythropoietin, which acts on HSCs to differentiate into embryonic red blood cells (rbc) (4). Hypoxia, in turn, is alleviated by the delivery of oxygen by rbc, which circulate in newly formed vessels. ...
Transport of oxygen by red blood cells (rbc) is critical for life and embryogenesis. Here, we determined that provision of the lipid mediator sphingosine 1-phosphate (S1P) to the systemic circulation is an essential function of rbc in embryogenesis. Mice with rbc-specific deletion of sphingosine kinases 1 and 2 (Sphk1 and Sphk2) showed embryonic lethality between E11.5 and E12.5 due to defects in vascular development. Administration of an S1P1 receptor agonist to pregnant dams rescued early embryonic lethality. Even though rbc-specific Sphk1 Sphk2-KO embryos were anemic, the erythropoietic capacity of hematopoietic stem cells (HSCs) was not impaired, suggesting that rbc can develop in the absence of sphingosine kinase activity. Indeed, transplantation of HSCs deficient for Sphk1 and Sphk2 into adult mice produced rbc that lacked S1P and attenuated plasma S1P levels in recipients. However, in adult animals, both rbc and endothelium contributed to plasma S1P. Together, these findings demonstrate that rbc are essential for embryogenesis by supplying the lysophospholipid S1P, which regulates embryonic vascular development via its receptors.
... Other critical factors in hematopoiesis are Rag1, which is essential for the differentiation of T and B lymphocytes (Petrie-Hanson et al., 2009), and Runx1, which regulates the formation of hematopoietic progenitors, myeloid cells and the vasculature (Jin et al., 2012;Kalev-Zylinska et al., 2002). The development of endothelial cells and vascular patterning is regulated by the vascular endothelial growth factor (VEGF) receptors Flt1 and Flk1 (Hirashima, 2009;Orkin and Zon, 1997;Siekmann et al., 2008), whereas the basic helix-loop-helix transcription factor Scl is crucial for both hematopoiesis and vasculogenesis (Gering et al., 1998). Although the molecules that regulate the formation of blood cells and blood vessels are well characterized, those involved in the specification of the hemangioblastic fate in ventral mesoderm are less well characterized. ...
Hematopoietic and vascular endothelial cells constitute the circulatory system and are both generated from the ventral mesoderm. However, the molecules and signaling pathways involved in ventral mesoderm formation and specification remain unclear. We found that zebrafish etv5a was expressed in the ventral mesoderm during gastrulation. Knockdown of Etv5a using morpholinos increased the proliferation of ventral mesoderm cells and caused defects in hematopoietic derivatives and in vascular formation. In contrast, the formation of other mesodermal derivatives, such as pronephros, somites and the gut wall, was not affected. Knockdown specificity was further confirmed by over-expression of an etv5a construct lacking its acidic domain. In conclusion, our data reveal that etv5a is essential for the inhibition of ventral mesoderm cell proliferation and for the formation of the hemato-vascular lineage.
... In addition to the EPO-EPOR-JAK2 system, a number of genes have been involved in regulating erythropoiesis, which include a variety of transcription factors (9,10), c-Kit tyrosine kinase (11) and various apoptosis regulators such as BclXL (12) FADD (13), and caspase-8 (14). We have recently demonstrated that CIS3 acts as a negative regulator of fetal liver erythropoiesis (15). ...
The cytokine-inducible SH2 protein-3 (CIS3/SOCS-3/SSI-3) has been shown to inhibit the JAK/STAT pathway and act as a negative
regulator of fetal liver erythropoiesis. Here, we studied the molecular mechanisms by which CIS3 regulates the erythropoietin
(EPO) receptor (EPOR) signaling in erythroid progenitors and Ba/F3 cells expressing the EPOR (BF-ER). CIS3 binds directly
to the EPOR as well as JAK2 and inhibits EPO-dependent proliferation and STAT5 activation. We have identified the region containing
Tyr401 in the cytoplasmic domain of the EPOR as a direct binding site for CIS3. Deletion of the Tyr401 region of the EPOR reduced the inhibitory effect of CIS3, suggesting that binding of CIS3 to the EPOR augmented the negative
effect of CIS3. Both N- and C-terminal regions adjacent to the SH2 domain of CIS3 were necessary for binding to EPOR and JAK2.
In the N-terminal region of CIS3, the amino acid Gly45 was critical for binding to the EPOR but not to JAK2, while Leu22 was critical for binding to JAK2. The mutation of G45A partially reduced ability of CIS3 to inhibit EPO-dependent proliferation
and STAT5 activation, while L22D mutant CIS3 was completely unable to suppress EPOR signaling. Moreover, overexpression of
STAT5, which also binds to Tyr401, reduced the binding of CIS3 to the EPOR, and the inhibitory effect of CIS3 against EPO signaling, while it did not affect
JAB/SOCS-1/SSI-1. These data demonstrate that binding of CIS3 to the EPOR augments the inhibitory effect of CIS3. CIS3 binding
to both EPOR and JAK2 may explain a specific regulatory role of CIS3 in erythropoiesis.
... In summary, our study provides definitive evidence that ankyrin and band 3 are not required for mouse fetal liver erythroblast enucleation. The hypothesis that mammalian erythroid membrane and cytoskeleton proteins are involved in enucleation is based on the fact that chromatin condensation occurs in all vertebrates but that only mammalian erythroblasts undergo nuclear extrusion [2,17]. During erythropoiesis, the membrane and cytoskeleton proteins undergo extensive reorganization. ...
During late stages of mammalian erythropoiesis the nucleus undergoes chromatin condensation, migration to the plasma membrane, and extrusion from the cytoplasm surrounded by a segment of plasma membrane. Since nuclear condensation occurs in all vertebrates, mammalian erythroid membrane and cytoskeleton proteins were implicated as playing important roles in mediating the movement and extrusion of the nucleus. Here we use erythroid ankyrin deficient and band 3 knockout mouse models to show that band 3, but not ankyrin, plays an important role in regulating the level of erythroid cell membrane proteins, as evidenced by decreased cell surface expression of glycophorin A in band 3 knockout mice. However, neither band 3 nor ankyrin are required for enucleation. These results demonstrate that mammalian erythroblast enucleation does not depend on the membrane integrity generated by the ankyrin-band 3 complex.
... Since PECAM-1 and CD41 did not discriminate between blood and endothelial cells at headfold stages, we chose to examine a different combination of markers to distinguish between the blood and vessel populations. GATA1 is required for erythroid development (Orkin and Zon, 1997) and is co-expressed with VE-Cadherin in blood island cells at headfold stages, a subset of which displayed hemangioblast properties (Yokomizo et al., 2007). ...
The mouse posterior primitive streak at neural plate/headfold stages (NP/HF, ~7.5 dpc-8 dpc) represents an optimal window from which hemangioblasts can be isolated. We performed immunohistochemistry on this domain using established monoclonal antibodies for proteins that affect blood and endothelial fates. We demonstrate that HoxB4 and GATA1 are the first set of markers that segregate independently to endothelial or blood populations during NP/HF stages of mouse embryonic development. In a subset of cells, both proteins are co-expressed and immunoreactivities appear mutually excluded within nuclear spaces. We searched for this particular state at later sites of hematopoietic stem cell emergence, viz., the aorta-gonad-mesonephros (AGM) and the fetal liver at 10.5-11.5 dpc, and found that only a rare number of cells displayed this character. Based on this spatial-temporal argument, we propose that the earliest blood progenitors emerge either directly from the epiblast or through segregation within the allantoic core domain (ACD) through reduction of cell adhesion and pSmad1/5 nuclear signaling, followed by a stochastic decision toward a blood or endothelial fate that involves GATA1 and HoxB4, respectively. A third form in which binding distributions are balanced may represent a common condition shared by hemangioblasts and HSCs. We developed a heuristic model of hemangioblast maturation, in part, to be explicit about our assumptions.
... The zebrafish embryo has long been recognized as an excellent model system for molecular-genetic analysis of vertebrate embryonic development (Detrich et al., 1999), one whose advantages complement, and perhaps exceed, those of the mouse (Orkin and Zon, 1997). Forward genetic screens using large-scale zygotic (Driever et al., 1996;Haffter et al., 1996), maternal (Pelegri and Mullins, 2004), and numerous targeted strategies have generated thousands of mutations in the zebrafish that affect all levels of development. ...
The large and transparent cells of cleavage-stage zebrafish embryos provide unique opportunities to study cell division and cytoskeletal dynamics in very large animal cells. Here, we summarize recent progress, from our laboratories and others, on live imaging of the microtubule and actin cytoskeletons during zebrafish embryonic cleavage. First, we present simple protocols for extending the breeding competence of zebrafish mating ensembles throughout the day, which ensures a steady supply of embryos in early cleavage, and for mounting these embryos for imaging. Second, we describe a transgenic zebrafish line [Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1] that expresses the green fluorescent protein (GFP)-labeled microtubule-binding part of ensconsin (EMTB-3GFP). We demonstrate that the microtubule-based structures of the early cell cycles can be imaged live, with single microtubule resolution and with high contrast, in this line. Microtubules are much more easily visualized using this tagged binding protein rather than directly labeled tubulin (injected Alexa-647-labeled tubulin), presumably due to lower background from probe molecules not attached to microtubules. Third, we illustrate live imaging of the actin cytoskeleton by injection of the actin-binding fragment of utrophin fused to GFP. Fourth, we compare epifluorescence-, spinning-disc-, laser-scanning-, and two-photon-microscopic modalities for live imaging of the microtubule cytoskeleton in early embryos of our EMTB-3GFP-expressing transgenic line. Finally, we discuss future applications and extensions of our methods.
... Erythropoiesis occurs in two distinct phases in zebrafish, primitive and definitive, which differ both temporally and spatially (see Orkin and Zon, 1997). In addition to the apparent lack of definitive erythrocytes, there was evidence of a decreased number of primitive erythrocytes at 36 hpf in retene-exposed zebrafish embryos {Supplemental video: bloodflow_36hpf.mov}. ...
In the embryo-larval stages of fish, alkylphenanthrenes such as retene (7-isopropyl-1-methylphenanthrene) produce a suite of developmental abnormalities typical of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including pericardial and yolk sac edema, cardiovascular dysfunction, and skeletal deformities. To investigate the mechanism and target tissue of retene toxicity, we used observational, histological, and protein knockdown techniques in zebrafish (Danio rerio) embryos. The primary overt signs of toxicity are pericardial edema and reduced blood flow, first observed at 36 h post-fertilization (hpf). The most pronounced effects at this stage are a reduced layer of cardiac jelly in the atrium and reduced diastolic filling. Conversely, an increased layer of cardiac jelly is observed at 72 hpf in retene-exposed embryos. Induction of cytochrome P4501A (CYP1A) is apparent in a subset of cardiomyocytes by 48 hpf suggesting that early cardiac effects may be due to AhR activation in the myocardium. Myocardial CYP1A induction is transient, with only endocardial induction observed at 72 hpf. Knockdown of cyp1a by morpholino oligonucleotides does not affect retene toxicity; however, ahr2 knockdown prevents toxicity. Thus, the mechanism of retene cardiotoxicity is AhR2-mediated and CYP1A-independent, similar to TCDD; however, the onset and proximate signs of retene toxicity differ from those of TCDD. Retene cardiotoxicity also differs mechanistically from the cardiac effects of non-alkylated phenanthrane, illustrating that alkyl groups can alter toxic action. These findings have implications for understanding the toxicity of complex mixtures containing alkylated and non-alkylated polycyclic aromatic hydrocarbons.
... For instance, EP 15 (generality = 1.44, 6 tissues) was significantly enriched for genes involved in erythropoiesis and was used primarily by adult bone marrow from both species and human fetal liver. Interestingly , the fetal liver is known to be critical for erythropoiesis during embryonic development, after which bone marrow becomes the predominant organ involved in this process [52] . Another example in this category includes EP 73 (generality = 0.93, 6 tissues), which was used by the kidney and liver in both species, and was enriched for genes involved in oxidative metabolism and gluconeogenesis. ...
An important research problem in computational biology is theidentification of expression programs, sets of co-activatedgenes orchestrating physiological processes, and thecharacterization of the functional breadth of these programs. Theuse of mammalian expression data compendia for discovery of suchprograms presents several challenges, including: 1) cellularinhomogeneity within samples, 2) genetic and environmental variationacross samples, and 3) uncertainty in the numbers of programs andsample populations. We developed GeneProgram, a new unsupervisedcomputational framework that uses expression data to simultaneouslyorganize genes into overlapping programs and tissues into groups toproduce maps of inter-species expression programs, which are sortedby generality scores that exploit the automatically learnedgroupings. Our method addresses each of the above challenges byusing a probabilistic model that: 1) allocates mRNA to differentexpression programs that may be shared across tissues, 2) ishierarchical, treating each tissue as a sample from a population ofrelated tissues, and 3) uses Dirichlet Processes, a non-parametricBayesian method that provides prior distributions over numbers ofsets while penalizing model complexity. Using real gene expressiondata, we show that GeneProgram outperforms several popularexpression analysis methods in recovering biologically interpretablegene sets. From a large compendium of mouse and human expressiondata, GeneProgram discovers 19 tissue groups and 100 expressionprograms active in mammalian tissues. Our method automaticallyconstructs a comprehensive, body-wide map of expression programs andcharacterizes their functional generality. This map can be used forguiding future biological experiments, such as discovery of genesfor new drug targets that exhibit minimal "cross-talk" withunintended organs, or genes that maintain general physiologicalresponses that go awry in disease states. Further, our method isgeneral, and can be applied readily to novel compendia of biologicaldata.
... By contrast, the adjacent pax2aexpressing pronephric cells were not affected in ints5 morphants ( Fig. 2A,B) (Majumdar et al., 2000). The expression of gata1, which is crucial for the specification of erythrocytes, was also reduced in ints5 morphants (Fig. 1P) (Orkin and Zon, 1997). This reduction is rescued by the co-injection of ints5 RNA (Fig. 1M,N,Q), whereas overexpression of ints5 RNA by itself does not affect scl or gata1 expression (Fig. 1K,L; data not shown). ...
Hematopoiesis, the dynamic process of blood cell development, is regulated by the activity of the bone morphogenetic protein (BMP) signaling pathway and by many transcription factors. However, the molecules and mechanisms that regulate BMP/Smad signaling in hematopoiesis are largely unknown. Here, we show that the Integrator complex, an evolutionarily conserved group of proteins, functions in zebrafish hematopoiesis by modulating Smad/BMP signaling. The Integrator complex proteins are known to directly interact with RNA polymerase II to mediate 3' end processing of U1 and U2 snRNAs. We have identified several subunits of the Integrator complex in zebrafish. Antisense morpholino-mediated knockdown of the Integrator subunit 5 (Ints5) in zebrafish embryos affects U1 and U2 snRNA processing, leading to aberrant splicing of smad1 and smad5 RNA, and reduced expression of the hematopoietic genes stem cell leukemia (scl, also known as tal1) and gata1. Blood smears from ints5 morphant embryos show arrested red blood cell differentiation, similar to scl-deficient embryos. Interestingly, targeting other Integrator subunits also leads to defects in smad5 RNA splicing and arrested hematopoiesis, suggesting that the Ints proteins function as a complex to regulate the BMP pathway during hematopoiesis. Our work establishes a link between the RNA processing machinery and the downstream effectors of BMP signaling, and reveals a new group of proteins that regulates the switch from primitive hematopoietic stem cell identity and blood cell differentiation by modulating Smad function.
... genes can be divided into two groups on the basis of sequence homology and expression pattern. The members of the first group, gata1, gata2, and gata3 have unique roles in hematopoiesis (for review, see Orkin and Zon 1997), whereas gata4, gata5, and gata6 are expressed in the ALPM and endoderm (for review, see Evans 1997). Studies of cis-regulatory elements have suggested that gata4, gata5, and gata6 have important roles in promoting the expression of both myocardial and endodermal genes. ...
The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study. Here we present evidence from both loss- and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development. We demonstrate that zebrafish Gata5 is encoded by the faust locus. Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors and the expression of normal levels of several myocardial genes including nkx2.5. Later, Gata5 is necessary for the elaboration of ventricular tissue. We further demonstrate that Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis. Significantly, overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial tissue. Together, these results implicate zebrafish Gata5 in controlling the growth, morphogenesis, and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5.
... Since MDS is a clonal hematopoietic stem cell disease characterized by ineffective hematopoiesis and peripheral cytopenias, TERT deficiency in zebrafish possibly recapitulates this disorder. During zebrafish hematopoiesis, definitive progenitors can be observed in the posterior ICM and in the dorsal aorta as early as 24 hpf [79,80,81]. However, since it is unknown whether zebrafish hematopoietic stem cells continue to arise de novo, we cannot rule out the possibility that a failure of later arising stem cell populations to develop may lead to the actual blood cell reduction which we observe by 72 hpf. ...
Although it is clear that telomerase expression is crucial for the maintenance of telomere homeostasis, there is increasing evidence that the TERT protein can have physiological roles that are independent of this central function. To further examine the role of telomerase during vertebrate development, the zebrafish telomerase reverse transcriptase (zTERT) was functionally characterized. Upon zTERT knockdown, zebrafish embryos show reduced telomerase activity and are viable, but develop pancytopenia resulting from aberrant hematopoiesis. The blood cell counts in TERT-depleted zebrafish embryos are markedly decreased and hematopoietic cell differentiation is impaired, whereas other somatic lineages remain morphologically unaffected. Although both primitive and definitive hematopoiesis is disrupted by zTERT knockdown, the telomere lengths are not significantly altered throughout early development. Induced p53 deficiency, as well as overexpression of the anti-apoptotic proteins Bcl-2 and E1B-19K, significantly relieves the decreased blood cells numbers caused by zTERT knockdown, but not the impaired blood cell differentiation. Surprisingly, only the reverse transcriptase motifs of zTERT are crucial, but the telomerase RNA-binding domain of zTERT is not required, for rescuing complete hematopoiesis. This is therefore the first demonstration of a non-canonical catalytic activity of TERT, which is different from "authentic" telomerase activity, is required for during vertebrate hematopoiesis. On the other hand, zTERT deficiency induced a defect in hematopoiesis through a potent and specific effect on the gene expression of key regulators in the absence of telomere dysfunction. These results suggest that TERT non-canonically functions in hematopoietic cell differentiation and survival in vertebrates, independently of its role in telomere homeostasis. The data also provide insights into a non-canonical pathway by which TERT functions to modulate specification of hematopoietic stem/progenitor cells during vertebrate development. (276 words).
... embryos had comparable total numbers of haematopoietic precursors within the yolk sac and the splanchnopleura (not shown). Moreover, using a cocktail of different cytokines and stromal cell lines in a clonal in vitro differentiation assay (Cumano et al., 1993; Ray et al., 1996), sek1 −/− and sek1 +/− precursors derived from the yolk sac and the splanchnopleura of E10.5 embryos were stimulated to develop into macrophages/monocytes, mast cells (not shown), and immunoglobulin-secreting B lymphocytes (sek1 +/+ IgM [ 1995; Orkin and Zon, 1997). Whereas haematopoiesis (as judged by colony formation from the yolk sac and the growth of mast cells, B cells and macrophages/monocytes) and vasculogenesis (as assessed by the results from sek1 −/− flk1 +/− lacZ mice) appear normal in sek1 −/− mice, these mutants had very small livers (Fig. 3A). ...
The stress signaling kinase SEK1/MKK4 is a direct activator of stress-activated protein kinases (SAPKs; also called Jun-N-terminal kinases, JNKs) in response to a variety of cellular stresses, such as changes in osmolarity, metabolic poisons, DNA damage, heat shock or inflammatory cytokines. We have disrupted the sek1 gene in mice using homologous recombination. Sek1(-/- )embryos display severe anemia and die between embryonic day 10.5 (E10.5) and E12.5. Haematopoiesis from yolk sac precursors and vasculogenesis are normal in sek1(-/- )embryos. However, hepatogenesis and liver formation were severely impaired in the mutant embryos and E11.5 and E12.5 sek1(-/- )embryos had greatly reduced numbers of parenchymal hepatocytes. Whereas formation of the primordial liver from the visceral endoderm appeared normal, sek1(-/-) liver cells underwent massive apoptosis. These results provide the first genetic link between stress-responsive kinases and organogenesis in mammals and indicate that SEK1 provides a crucial and specific survival signal for hepatocytes.
This book is a completely revised new edition of the definitive reference on disorders of hemoglobin. Authored by world-renowned experts, the book focuses on basic science aspects and clinical features of hemoglobinopathies, covering diagnosis, treatment, and future applications of current research. While the second edition continues to address the important molecular, cellular, and genetic components, coverage of clinical issues has been significantly expanded, and there is more practical emphasis on diagnosis and management throughout. The book opens with a review of the scientific underpinnings. Pathophysiology of common hemoglobin disorders is discussed next in an entirely new section devoted to vascular biology, the erythrocyte membrane, nitric oxide biology, and hemolysis. Four sections deal with α and β thalassemia, sickle cell disease, and related conditions, followed by special topics. The second edition concludes with current and developing approaches to treatment, incorporating new agents for iron chelation, methods to induce fetal hemoglobin production, novel treatment approaches, stem cell transplantation, and progress in gene therapy.
Zebrafish is a novel pharmacological screening model for increasing the pace of drug discovery and development. It is an extensively used vertebrate model with the purpose for understanding disease development as well as its progression. Zebrafish offers a variety of advantages over the conventional pharmacological models including economical maintenance, high fecundity, transparent body, and ease of inducing genetic mutations using a variety of techniques. Zebrafish have high similarity with humans and offer an alternative strategy for high-throughput screening of new chemical entities. Blood disorders constitute a significant number of human pathologies ranging from anemia to leukemia. To this end, zebrafish offers a unique platform to study a plethora of human hematological disorders. In the current chapter, we provide the details of advantages and limitations of using zebrafish in hematology research. The use of zebrafish in studying various non-malignant disorders like anemia, bone marrow failure, coagulation disorders, and immunodeficiency has been covered.
Further, the advantage of zebrafish serving as a model to understand
various malignant blood disorders like chronic lymphoblastic leukemia
(CLL), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia (CML) along with acute myeloid leukemia (AML) has been
described. In a nutshell, this chapter accentuates the role of zebrafish as an emerging pharmacological model to evaluate the pathology of
hematological disorders and development of drugs for the treatment thereof.
The insecticidal Cry toxins produced by Bacillus thuringiensis (Bt) are powerful tools for insect control. Cry toxin receptors such as cadherin (CAD), ABCC2 transporter and alkaline phosphatase (ALP), located on insect midgut cells, are needed for Cry toxicity. Although insect cell lines are useful experimental models for elucidating toxin action mechanism, most of them show low expression of Cry-receptors genes. The GATA transcription factor family plays important roles in regulating development and differentiation of intestine stem cells. Here, we investigated whether GATAs transcription factors are involved in the expression of Cry1Ac-receptors genes, using multiple insect cell lines. Four GATA genes were identified in the transcriptome of the midgut tissue from the lepidopteran larvae Helicoverpa armigera. These HaGATA genes were transiently expressed in three lepidopteran cell lines, Spodoptera frugiperda Sf9,H. armigera QB-Ha-E5 and Trichoplusia ni Hi5. Analysis of transcription activity using transcriptional gene-fusions showed that only H. armigera GATAe (HaGATAe) significantly increased the transcription of CAD, ABCC2 and ALP receptors genes in all insect cell lines. Key DNA regions for HaGATAe regulation were identified in the promoter sequence of these Cry-receptors genes by using promoter deletion mapping. The transient expression of HaGATAe in these cell lines, conferred sensitivity to Cry1Ac toxin, although in Hi5 cells the susceptibility to Cry1Ac was lower than in other two cell lines. High sensitivity to Cry1Ac correlated with simultaneous transcription of ABCC2 and CAD genes in Sf9 and QB-Ha-E5 cells. Our results reveal that HaGATAe enhances transcription of several lepidopteran Cry1Ac receptor genes in cultured insect cells.
The totipotent mouse embryonic stem (ES) cell is known to differentiate into cells expressing the β-globin gene when stimulated with bone morphogenetic protein (BMP)-4. Here, we demonstrate that BMP-4 is essential for generating both erythro-myeloid colony-forming cells (CFCs) and lymphoid (B and NK) progenitor cells from ES cells and that vascular endothelial growth factor (VEGF) synergizes with BMP-4. The CD45+ myelomonocytic progenitors and Ter119+ erythroid cells began to be detected with 0.5 ng/mL BMP-4, and their levels plateaued at approximately 2 ng/mL. VEGF alone weakly elevated the CD34+ cell population though no lymphohematopoietic progenitors were induced. However, when combined with BMP-4, 2 to 20 ng/mL VEGF synergistically augmented the BMP-4-dependent generation of erythro-myeloid CFCs and lymphoid progenitors from ES cells, which were enriched in CD34+ CD31lo and CD34+CD45− cell populations, respectively, in a dose-dependent manner. Furthermore, during the 7 days of in vitro differentiation, BMP-4 was required within the first 4 days, whereas VEGF was functional after the action of BMP-4 (in the last 3 days). Thus, VEGF is a synergistic enhancer for the BMP-4-dependent differentiation processes, and it seems to be achieved by the ordered action of the 2 factors.
Many experimental models have been used to study erythropoiesis. Even prior to the advent of the genetic manipulation of animal models, erythropoiesis was examined in the mouse, chicken, sheep, goat, and rabbit, among other vertebrates. Erythroid cell lines derived from human blood cancers were also very useful, as they could be genetically manipulated more easily than whole animals. Genetic models in the mouse, zebrafish, and frog have provided a plethora of information advancing our understanding of erythropoiesis, and remain gold standards in the field for studies of hemoglobin switching, and experiments to study authentic blood cell development. Mouse and human embryonic stem (ES) and induced pluripotent (iPS) cells can be differentiated to erythroid cells in culture, though their use is somewhat limited by their propensity to express only the embryonic and fetal globin genes. Some very useful cell lines have been developed by manipulating ES or fetal liver erythroid progenitor cells from knockout mouse models. In recent years, our understanding of erythropoiesis has improved, due to the ability to knock down genes in native human hematopoietic stem and progenitor cells derived from umbilical cord blood or bone marrow, and differentiate them ex vivo to the erythroid lineage. These native cells, and cell lines derived from them, are now providing essential information about human erythropoiesis, which is complementary to that obtained from animal studies. This review provides some perspective about the cell and animal models used to study erythropoiesis over the years.
The establishment of the hematopoietic system entails a series of developmental decisions, followed by expansion of immature progenitors or hematopoietic stem cells (HSCs) and the subsequent commitment of later progenitors to differentiation along selected lineages (1). Within the early embryo, ventral (or posterior) mesoderm gives rise to presumptive hemangioblasts (2) that are further specified to embryonic erythroid precursors and vascular cells in the developing yolk sac blood islands (Fig. 1). Later, intraembryonic hematopoiesis occurs in the fetal liver, most likely seeded from progenitors or HSCs located in the aortic/gonad/mesonephros (AGM) region (3–6). In addition to these important developmental decisions, amplification of hematopoietic progenitors within the yolk sac and embryonic compartments is necessary to provide the total number of cells required to meet increasing demands as the embryo grows. Various inductive events under the control of growth factors presumably lead to the origin of the hemangioblast, the specification of embryonic hematopoiesis, and the appearance of HSCs in the AGM region. The critical developmental decisions are thought to be executed by transcription factors, functioning in a combinatorial manner. These are the subject of this review. Genetic approaches have culminated in the identification of several transcription factors, or transcription factor associated proteins that are essential for various aspects of hematopoietic development. Examples are discussed below with the aim of defining some principles underlying hematopoietic development. The factors reviewed herein are summarized in Table 1 and are individually considered throughout the chapter.
A cell is considered to be totipotent if it displays the full range of developmental capabilities when it is placed in a permissive environment. The contributive definition of totipotency depends on the cellular environment, which can either enhance or depress the survival of test cells, leading to a selection process that may not necessarily be meaningful in terms of intrinsic cell potency. As organisms were generated through processes more complex than cleavage or budding, culminating in sexual reproduction, pluripotency became essential to generate a complete body. The germ line developed as a distinct cell lineage, enabling the genetic flow from one generation to the next. The isolation of pluripotent stem cells is an important achievement in biology and medicine because these cells have widespread implications for developmental biology, drug discovery and testing, and transplantation medicine. Stem cells can help in unraveling the complex events that occur during mammalian development, thereby identifying the factors involved in the cellular decision-making process that leads to cell specialization. Human embryonic stem (ES) cells, in particular, could offer insights into the developmental events that cannot be studied directly in humans in utero or fully understood using animal models. Drug testing on cultured human embryonic stem cells could also reduce the risk of drug-related birth defects. Pluripotent cell lines have been derived from the inner cell mass (ICM) of embryos at the blastocyst stage (ES cells) or isolated from aborted fetuses. SCNT is another method used to produce pluripotent stem cells. There are a wide variety of antigenic and molecular markers that—by virtue of being present in embryonic carcinoma (EC), ES, and epiblast cells—are believed to be specific to pluripotent cells.
Over the past 30 years we have become familiar with the way in which different types of hemoglobin are expressed at different stages of development. In the human embryo the main hemoglobins include Hb Portland (ζ2γ2), Hb Gower I (ζ2ε2), and Gower II (α2ε2). In the fetus, HbF (α2γ2) predominates and in the adult, HbA (α2β2) makes up the majority of hemoglobin in red cells. These simple facts belie the complexity of the cellular and molecular processes that bring about these beautifully coordinated changes in the patterns of globin gene expression throughout development. To understand these phenomena we have to consider the individual components including 1) the origins of erythroid cells in development, 2) the processes by which erythroid cells differentiate to mature red cells at each developmental stage, and 3) the molecular events that produce the patterns of gene expression we observe. Two different types of erythroid cells are observed during development. The first erythroid cells to be seen in the developing embryo are located in the blood islands of the yolk sac. These primitive erythroid cells are morphologically different from the definitive erythroid cells made in the fetal liver and bone marrow and contain predominantly embryonic hemoglobins. Somewhat later during embryonic development, definitive erythroid and other hematopoietic cells originate from multipotent cells identified in a part of the embryo that lies near the dorsal aorta, in the region close to where the kidneys first develop: the so-called aorta-gonads-mesonephros (AGM) region.
The endothelium, the cell layer that forms the inner lining of blood vessels, is a spatially distributed system that extends to all areas of the human body. Clinical and basic research demonstrates that the endothelium plays a crucial role in mediating homeostasis and is involved in virtually every disease, either as a primary determinant of pathophysiology or as a victim of collateral damage. The endothelium has remarkable, though largely untapped, diagnostic and therapeutic potential. This volume bridges the bench-to-bedside gap in endothelial biomedicine, advancing research and development and improving human health. The book is the first to systematically integrate knowledge about the endothelium from different organ-specific disciplines, including neurology, pulmonary, cardiology, gastroenterology, rheumatology, infectious disease, hematology-oncology, nephrology, and dermatology. It's interdisciplinary approach, which draws on expertise from such diverse fields as evolutionary biology, comparative biology, molecular and cell biology, mathematical modeling and complexity theory, translational research, and clinical medicine.
Introduction: Erythropoiesis involves the production of mature enucleated erythrocytes from committed erythroid progenitor cells, which in turn are derived from multilineage progenitors and ultimately from the hematopoietic stem cell (HSC). In human the mature erythrocytes turn over at a rate of approximately 1% per day and it can be estimated that maintaining the red blood cell count in an adult requires approximately 2.4 × 106 new erythrocytes to be produced each second. It is not surprising, therefore, that the regulation of erythropoiesis is a complex, multifaceted process that has to cope with not only maintaining the steady state but also with providing reserves to cope rapidly with increased demand as a result of physiological or pathological demands. In this chapter we will consider the developmental origins of red cell production, their differentiation from HSCs as well as production of the hormone erythropoietin. We will examine how erythropoietin responds to tissue hypoxia and exerts its effect through cell surface receptors on erythroid cells to trigger a number of cell signaling cascades to maintain, through critical transcription factors, the survival, proliferation, and maturation of the erythron. Erythropoiesis During Development: The first erythrocytes appearing during vertebrate development are known as primitive erythrocytes. These cells are produced by a transient first wave of hematopoiesis, which is almost entirely dedicated to the production of primitive red cells. Primitive erythropoiesis has been studied in evolutionary distant vertebrates, in particular in fish, amphibians, birds, and mammals.
This chapter reviews transcriptional programs of stem cells with a particular, although not exclusive, emphasis on stem and progenitor cells of the hemopoietic system. In the context of transplantation, stem cell definitions are more stringent and the standard used is the ability to reconstitute an entire tissue system and maintain it for an extended period, and preferably, for the lifetime of the organism. Stem cells in adult muscle may function in repair, as may stem cells in the liver, kidney, pancreas, and the central nervous system. These cells, derived from the inner cell mass of in vitro fertilization (IVF) embryos surplus to requirements exhibit pluripotent differentiation in vitro. A classic early example of this type is the so-called brains to blood study in which neurosphere-derived neural stem cells (NSCs) were reported to contribute to hemopoiesis in a murine transplantation model thereby crossing not only tissue but also germ layer boundaries.
Significance
Mouse models have been instrumental in advancing our understanding of blood cell production. Although many studies have suggested specific differences between human and mouse red cell production (erythropoiesis), a global study of such similarities and differences has been lacking. By computationally comparing global gene expression data from adult human and mouse erythroid precursors representing the distinct stages of maturation, we showed that, while the overall transcriptional landscape has changed, critical erythroid gene signatures and transcriptional regulators have remained conserved. Importantly, these analyses can serve as a tool to integrate data between human and mouse erythropoiesis research, explain why certain human blood diseases are not faithfully recapitulated in mouse models, and highlight hurdles in translating therapeutic findings from mice to humans.
The zebrafish vascular and hematopoietic systems are similar in many ways to those of all other vertebrates, including mammals. The study of genes that play important roles in zebrafish vasculogenesis and hematopoiesis is facilitated by many of the unique attributes that the zebrafish offers as a model system. The transparent nature of zebrafish embryos makes it possible to observe blood cells circulating throughout the vasculature from the onset of circulation until at least the end of the larval stage. Genetic approaches can be used to identify mutations that affect virtually all aspects of zebrafish development, including blood vessel formation and hematopoiesis. Screens for mutations that affect these processes are further enhanced by the fact that zebrafish embryos can survive a number of days without a functional cardiovascular system, thus making it possible to study both the early and late effects of vascular and hematopoietic mutations. This characteristic is an important advantage not offered by, for example, the mouse embryo. Combined together, these attributes have propelled the zebrafish forward as a useful model system for the study of these critical processes.
Phenotype-driven approaches to gene discovery using inbred mice have been instrumental in identifying genetic determinants
of inherited blood dyscrasias. The recessive mutant scat (severe combined anemia and thrombocytopenia) alternates between crisis and remission episodes, indicating an aberrant regulatory
feedback mechanism common to erythrocyte and platelet formation. Here, we identify a missense mutation (G125V) in the scat Rasa3 gene, encoding a Ras GTPase activating protein (RasGAP), and elucidate the mechanism producing crisis episodes. The mutation
causes mislocalization of RASA3 to the cytosol in scat red cells where it is inactive, leading to increased GTP-bound Ras. Erythropoiesis is severely blocked in scat crisis mice, and ∼94% succumb during the second crisis (∼30 d of age) from catastrophic hematopoietic failure in the spleen
and bone marrow. Megakaryopoiesis is also defective during crisis. Notably, the scat phenotype is recapitulated in zebrafish when rasa3 is silenced. These results highlight a critical, conserved, and nonredundant role for RASA3 in vertebrate hematopoiesis.
Lymphoid cellsMyeloid lineageErythroid cellsMegakaryocyte lineage cells
Hematopoietic development in the mammal can be represented as a numerically expanding hierarchy of cell populations that are progressively restricted in their self-renewal and differentiation abilities. Classical functional studies have now been extebded to provide exact physical descriptions of various stages in the Hematopoietic hierarchy. In particular, much information is available that defines the properties of the most primitive stem cell compartment. In addition, a number of in vitro culture systems suggest the possibility of maintaining and expanding these cells in a defined context. In all developmental systems, unique profiles of expressed genes define distinct differentiation stages. Within these profiles are gene products that play crucial roles in the regulation of cell-fate decisions. Recent progress in hematopoietic biology provides the framework within which to define molecular phenotypes for hematopoietic stem cells and their immediate clonal progeny. Identifying novel gene products expressed predominantly in uncommittes stem cells together with functional loos and gain-of-function approaches should begin to unravel the molecular mechanisms that govern biological phenomena such as self-renewal, commitment, and proliferation in the hematopoietic system.
The view that all blood derives from ventral mesoderm has been challenged in recent years. In the Xenopus embryo, it is now clear that the embryonic blood compartment, the ventral blood island (VBI), is derived from regions of
the pre-gastrula embryo traditionally referred to as dorsal as well as ventral. Furthermore, recent lineage labelling studies
in Xenopus, show that the adult blood lineage in the dorsal lateral plate (DLP) mesoderm arises independendy of the embryonic lineage.
Thus, there appear to be three distinct sources of blood in Xenopus embryos, two giving rise to the VBI and one the DLP. Distinct origins coupled with separate migration pathways through the
embryo suggest that the three populations may be independendy programmed during development. Perturbation of BMP signalling
shows that all three require this signal in order to form the putative bipotential precursor of blood and endothelium, the
hemangioblast. Differences between the embryonic populations and the adult lineage however have been detected with respect
to retinoid signalling during gastrulation, and also with respect to specific gene responses to BMP signalling. Experimental
manipulations of this model system are beginning to inform our understanding of the developmental programming of hematopoietic
stem cells.
Recent studies have shown that hematopoietic transcription factors can engage in multiple protein-protein interactions. Accumulating evidence indicates that specific complexes define differentiation lineages and differentiation stages. It is proposed that these complexes acquire new functions during blood cell differentiation through successive changes in composition — much as discussion topics of groups at a cocktail party take new directions as new people join and others leave.
Recent studies have identified the transcription factor Pax5 as a critical determinant of commitment to the B-lymphocyte pathway. Surprisingly, Pax5 appears to achieve this primarily through suppressing alternative haematopoietic lineage fates.
The neural crest is a transient, multipotent cell population in the developing vertebrate embryo that migrates extensively and contributes to a staggering diversity of cell lineages. Neural crest progenitors are specified at the neural plate border during gastrulation; however, commitment to the neural crest lineage does not occur for some time. I find that the chick neural plate border is characterized by co-expression of many neural crest specifier genes, previously considered “late” signals, which often overlap with “early” neural plate border genes. This suggested that continuously expressed members of the neural crest gene regulatory network may be modulated or repressed for proper maintenance of the multipotent state. Consistent with this possibility, several members of the Polycomb Group of epigenetic repressors are expressed at these early stages. For example, the stem cell factor Bmi-1 is expressed at the neural plate border, dorsal neural folds, and migrating neural crest, but is extinguished in differentiated derivatives. Morpholino-mediated knock-down of Bmi-1 causes early upregulation of Msx1, FoxD3, and Sox9 in the chick neurula without affecting cell proliferation. Conversely, Bmi-1 over-expression causes a downregulation of Msx1, suggesting that it negatively regulates neural crest network genes. I find that several alternative splice variants of Bmi-1 are expressed in the developing chick and that an N-terminal variant, V4, acts as a dominant-negative regulator of the full-length version, up-regulating Msx1 expression. Taken together, these results suggest that neural crest progenitors are exposed to numerous specification signals during gastrulation, some of which are regulated by Polycomb Group factors such as Bmi-1. The activity of Bmi-1, in turn, is modulated by alternatively spliced variants, demonstrating an additional level of regulatory complexity acting during early neural crest development.
Erythropoiesis is regulated such that a sufficient number of mature erythrocytes is produced. Down-regulation of erythropoiesis causes various types of anemia. Although some anemia-related genes have been identified, there are several types of anemic disease for which the molecular mechanisms are yet unclear, suggesting that unidentified genes in addition to the classical cytokine pathways play important roles in anemia. To address this issue, a new animal model for anemia is required. We established a reversible anemic model in zebrafish by keeping fish at 17 degrees C, a low water temperature. In zebrafish kidney marrow, expression of several genes encoding hematopoietic transcription factors (Runx1, scl, c-myb and GATA-2) and particularly erythropoiesis-related factors (klfd, hbaa1, ba1, GATA-1, EPO, and EPOr) was down-regulated, whereas myelopoiesis-related factors (csf1a and csf3) was up-regulated in low temperature conditions. We propose that this zebrafish model is useful to identify novel genes for hematopoiesis, particularly erythropoiesis.
Significant advances in the use of genetic and molecular biology strategies have recently begun to identify genes that have a major impact on the determination, commitment and developmental potential of hematopoietic stem cells. Using a variety of experimental strategies, genes such as SCL, GATA-2, HoxB4, Flk-2, c-mpl, dlk, and others have been implicated as important regulators of stem cell growth. In addition, genetic mapping has identified several loci that correlate strongly with stem cell numbers and proliferation.
The CBFβ subunit is the non-DNA-binding subunit of the heterodimeric core-binding factor (CBF). CBFβ associates with DNA-binding CBFα subunits and increases their affinity for DNA. Genes encoding the CBFβ subunit (CBFB) and one of the CBFα subunits (CBFA2, otherwise known as AML1) are the most frequent targets of chromosomal translocations in acute leukemias in humans. We and others previously demonstrated that homozygous disruption of the mouse Cbfa2 (AML1) gene results in embryonic lethality at midgestation due to hemorrhaging in the central nervous system and blocks fetal liver hematopoiesis. Here we demonstrate that homozygous mutation of the Cbfb gene results in the same phenotype. Our results demonstrate that the CBFβ subunit is required for CBFα2 function in vivo.
We have investigated the role of erythroid Kruppel-like factor (EKLF) in expression of the human beta-globin genes in compound EKLF knockout/human beta-locus transgenic mice. EKLF affects only the adult mouse beta-globin genes in homozygous knockout mice; heterozygous mice are unaffected. Here we show that EKLF knockout mice express the human epsilon and gamma-globin genes normally in embryonic red cells. However, fetal liver erythropoiesis, which is marked by a period of gamma- and beta-gene competition in which the genes are alternately transcribed, exhibits an altered ratio of gamma- to beta-gene transcription. EKLF heterozygous fetal livers display a decrease in the number of transcriptionally active beta genes with a reciprocal increase in the number of transcriptionally active gamma genes. beta-Gene transcription is absent in homozygous knockout fetuses with coincident changes in chromatin structure at the beta promoter. There is a further increase in the number of transcriptionally active gamma genes and accompanying gamma gene promoter chromatin alterations. These results indicate that EKLF plays a major role in gamma- and beta-gene competition and suggest that EKLF is important in stabilizing the interaction between the Locus Control Region and the beta-globin gene. In addition, these findings provide further evidence that developmental modulation of globin gene expression within individual cells is accomplished by altering the frequency and/or duration of transcriptional periods of a gene rather than changing the rate of transcription.
Previously, we showed that the promoter of the gene encoding preproendothelin-1 (PPET-1) contains a GATA motif that is essential for activity and interacts with a nuclear factor similar in size and binding specificity to the erythroid transcription factor GATA-1. To identify this endothelial GATA-binding protein, a human endothelial cell cDNA library was screened with oligonucleotide probes for a portion of the zinc finger domain of GATA-1. A 2.6-kilobase cDNA encoding a 470 amino acid protein was obtained. Sequence analysis revealed a predicted protein which is the human counterpart of a related chicken protein, designated GATA-2. Human GATA-2 is expressed by a variety of cells, including erythroid, HeLa, and endothelial cells. A complex of a GATA-containing probe and recombinant GATA-2 expressed in COS cells comigrates with that present in gel shift experiments with nuclear extract derived from endothelial cells. In addition, expressed human GATA-2 protein transactivates reporter gene constructs containing either minimal GATA promoter elements or the native PPET-1 promoter in a cotransfection assay. Retinoic acid treatment of endothelial cells results in down-regulation of GATA-2 expression as well as down-regulation of PPET-1 gene expression. Human homologs of other known GATA-binding transcription factors are either absent from endothelial cells (in the case of GATA-1) or made in small quantities and not significantly affected by retinoid acid in these cells (in the case of GATA-3), making it unlikely that they regulate the PPET-1 gene. We propose that GATA-2 is the GATA-binding protein required for PPET-1 gene expression in endothelial cells.
GATA-1, a transcription factor of the 'zinc-finger' family, is required for the development of mature erythroid cells and is also highly expressed in the megakaryocytic and mast cell lineages. The helix-loop-helix gene SCL (or TAL) is expressed in the same three hematopoietic lineages as GATA-1. To explore the role of GATA-1 and SCL in hematopoietic differentiation, we introduced a new expression vector bearing each gene into the early myeloid cell line 416B, which could originally differentiate in vivo along the megakaryocytic and granulocytic lineages. Enforced expression of SCL at high levels did not provoke differentiation, but GATA-1 induced the appearance of megakaryocytes as assessed by morphology, the presence of acetylcholinesterase and a polyploid DNA content. Although GATA-1 is thought to stimulate its own transcription in erythrocytes, expression of the endogenous gene was not increased in the megakaryocytic lines; hence GATA-1 may not be autoregulatory in this lineage. Megakaryocytic differentiation was accompanied by a marked decrease in the myeloid surface marker Mac-1. The absence of mast cell or erythroid differentiation suggests that GATA-1 may not be sufficient to provoke maturation along these lineages or that these pathways are impeded in 416B cells. These results demonstrate that a member of the GATA gene family can act as an important regulator of megakaryocytic differentiation.
The SCL (tal-1, TCL5) gene is a member of the basic domain, helix-loop-helix (bHLH) class of putative transcription factors. We found that (i) the SCL promoter for exon Ia contains a potential recognition site for GATA-binding transcription factors, (ii) SCL mRNA is expressed in all erythroid tissues and cell lines examined, and (iii) SCL mRNA increases upon induced differentiation of murine erythroleukemia (MEL) cells, and inferred that SCL may play a physiologic role in erythroid differentiation. We used gel shift and transfection assays to demonstrate that the GATA motif in the SCL promoter binds GATA-1 (and GATA-2), and also mediates transcriptional transactivation. To identify a role for SCL in erythroid differentiation, we generated stable transfectants of MEL and K562 (a human chronic myelogenous leukemia cell line that can differentiate along the erythroid pathway) cells overexpressing wild-type, antisense or mutant SCL cDNA. Increasing the level of SCL expression in two independent MEL lines (F4-6 and C19, a 745 derivative) and K562 cells increased the rate of spontaneous (i.e. in the absence of inducer) erythroid differentiation. Conversely, induced differentiation was inhibited in MEL transfectants expressing either antisense SCL cDNA or a mutant SCL lacking the basic domain. Our experiments suggest that the SCL gene can be a target for the erythroid transcription factor GATA-1 and that the SCL gene product serves as a positive regulator of erythroid differentiation.
The human SCL gene is a member of the family of genes that encode the helix-loop-helix (HLH) class of DNA-binding proteins. A murine SCL cDNA was isolated from a normal macrophage cDNA library by using HLH-specific oligonucleotides as hybridization probes. The coding region is 987 base pairs and encodes a predicted protein of 34 kDa. The nucleotide sequence of the coding region shows 88% identity to the human SCL gene, and the amino acid sequence is 94% identical. The HLH motif and upstream hydrophilic region are entirely conserved in the murine and human proteins. The identity between the mouse and human sequences was less marked in the 5' and 3' untranslated regions. Two murine SCL transcripts that differ in the 3' noncoding region have been detected in fetal liver and various cell lines. Variation was also observed in the 5' untranslated region. Interestingly, immediately downstream of the protein-termination codon, both the human SCL sequence and the murine homolog share an E-box element--the suggested target site for DNA binding of HLH proteins. The murine SCL homolog was mapped to the central part of chromosome 4.
The c-myb proto-oncogene encodes a sequence-specific DNA-binding protein. To better understand its normal biological function, we have altered the c-myb gene by homologous recombination in mouse embryonic stem cells. Resulting homozygous c-myb mutant mice displayed an interesting phenotype. At day 13 of gestation these mice appeared normal, suggesting that c-myb is not essential for early development. By day 15, however, the mutant mice were severely anemic. Analysis indicated that embryonic erythropoiesis, which occurs in the yolk sac, was not impaired by the c-myb alteration. Adult-type erythropoiesis, which first takes place in the fetal liver, was greatly diminished in c-myb mutants, however. Additional hematopoietic lineages were similarly affected. These results are compatible with a role for c-myb in maintaining the proliferative state of hematopoietic progenitor cells.
A chromosomal translocation in a T-cell leukemia involving the short arm of human chromosome 11 at band 11p15 disrupts the rhombotin gene. This gene encodes a protein with duplicated cysteine-rich regions called LIM domains, which show homology to zinc-binding proteins and to iron-sulfur centers of ferredoxins. Two homologues of the rhombotin gene have now been isolated. One of these, designated Rhom-2, is located on human chromosome 11 at band 11p13, where a cluster of T-cell leukemia-specific translocations occur; all translocation breakpoints at 11p13 are upstream of the Rhom-2 gene. Human and mouse Rhom-2 are highly conserved and, like rhombotin, encode two tandem cysteine-rich LIM domains. Rhom-2 mRNA is expressed in early mouse development in central nervous system, lung, kidney, liver, and spleen but only very low levels occur in thymus. The other gene, designated Rhom-3, is not on chromosome 11 but also retains homology to the LIM domain of rhombotin. Since the Rhom-2 gene is such a common site of chromosomal damage in T-cell tumors, the consistency of translocations near the rhombotin gene was further examined. A second translocation adjacent to rhombotin was found and at the same position as in the previous example. Therefore, chromosome bands 11p15 (rhombotin) and 11p13 (Rhom-2) are consistent sites of chromosome translocation in T-cell leukemia, with the 11p15 target more rarely involved. The results define the rhombotin gene family as a class of T-cell oncogenes with duplicated cysteine-rich LIM domains.
The tal-1 gene is altered as a consequence of the t(1;14) (p32;q11) chromosome translocation observed in 3% of patients with T cell acute lymphoblastic leukemia (T-ALL). tal-1 encodes a helix-loop-helix (HLH) domain, a DNA binding and dimerization motif found in a number of proteins involved in cell growth and differentiation. We now report that an additional 25% of T-ALL patients bear tal-1 gene rearrangements that are not detected by karyotype analysis. These rearrangements result from a precise 90 kb deletion (designated tald) that arises independently in different patients by site-specific DNA recombination. Since the deletion junctions resemble the coding joints of assembled immunoglobulin genes, tald rearrangements are likely to be mediated by aberrant activity of the immunoglobulin recombinase. Moreover, t(1;14)(p32;q11) translocations and tald rearrangements disrupt the coding potential of tal-1 in an equivalent manner, and thereby generate a common genetic lesion shared by a significant proportion of T-ALL patients.
An erythroid specific, inducible enhancer associated with hypersensitive site II (HS II) plays a central role in the function
of the human β globin dominant control region. The HS II enhancer consists of tandem AP-1 binding sites and has been shown
to bind members of the ubiquitous jun and fos families of proteins. The same sites are now shown to bind the erythroid specific
protein, NF-E2. Inducibility of the HS II enhancer depends on NF-E2 binding, even in the presence of another hypersensitive
site. Further, increased activity of the enhancer in induced K562 cells correlates with the presence of NF-E2, which appears
to be present in a modified form. NF-E2 is distinct from some enhancer binding proteins in K562 nuclear extracts, in that
it does not contain Fos or Fra-1 protein. Thus, binding by NF-E2 may be the mechanism, whereby tandem AP-1 binding sites confer
erythroid specificity on the HS II enhancer.
We describe the structural organization of the human SCL gene, a helix-loop-helix family member which we believe plays a fundamental
role in hematopoietic differentiation. The SCL locus is composed of eight exons distributed over 16 kb. SCL shows a pattern
of expression quite restricted to early hematopoietic tissues, although in malignant states expression of the gene may be
somewhat extended into later developmental stages. A detailed analysis of the transcript(s) arising from the SCL locus revealed
that (i) the 5' noncoding portion of the SCL transcript, which resides within a CpG island, has a complex pattern of alternative
exon utilization as well as two distinct transcription initiation sites; (ii) the 5' portions of the SCL transcript contain
features that suggest a possible regulatory role for these segments; (iii) the pattern of utilization of the 5' exons is cell
lineage dependent; and (iv) all of the currently studied chromosomal aberrations that affect the SCL locus either structurally
or functionally eliminate the normal 5' transcription initiation sites. These data suggest that the SCL gene, and specifically
its 5' region, may be a target for regulatory interactions during early hematopoietic development.
The murine, erythroid DNA-binding protein GF-1 (also known as NF-E1, Eryf 1), a 413-amino acid polypeptide with two novel finger domains of the Cx-Cx variety, recognizes a consensus GATA motif present in cis elements of the majority of erythroid-expressed genes. We have performed a structure-function analysis of this protein to evaluate its potential as a transcriptional activator and to examine the role of the finger domains in DNA binding. Using a cotransfection assay, we find that GF-1 is a potent transcriptional activator with several activation domains but that this is revealed only in heterologous cells and with reporters containing minimal promoters onto which either a single or multiple GATA-binding sites are placed. The two fingers of GF-1 are functionally distinct and cooperate to achieve specific, stable DNA binding. The amino finger is necessary only for full specificity and stability of binding, whereas the carboxyl finger is required for binding. The role of each finger is more pronounced with some GATA-binding sites than with others, suggesting a diversity of interactions between GF-1 and different target sites. The complex activation and DNA-binding properties of GF-1 are likely to contribute to the ability of this single protein to participate widely in gene expression throughout erythroid development.
We have identified the human gene, SCL. We discovered this gene because of its involvement in a chromosomal translocation associated with the occurrence of a stem cell leukemia manifesting myeloid and lymphoid differentiation capabilities. Here we report the sequence of a cDNA for the normal SCL transcript, as well as for an aberrant fusion transcript produced in the leukemic cells. Although different at their 3' untranslated regions, both cDNAs predict a protein with primary amino acid sequence homology to the previously described amphipathic helix-loop-helix DNA binding and dimerization motif of the Ly1-1, myc, MyoD, immunoglobulin enhancer binding, daughterless, and achaete-scute families of genes. For these cDNAs, at least two different 5' ends are predicted, both of which retain this putative DNA binding domain and predict proteins in the range of 20-30 kDa. SCL mRNA is observed in "early" hematopoietic tissues. Taken together, these studies lead to the speculation that SCL plays a role in differentiation and/or commitment events during hematopoiesis.
We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprintng,
gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation,
contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor,
which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of
the human β-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the ApI consensus,
it appears to be different from ApI.
Systematic genome-wide mutagenesis screens for embryonic phenotypes have been instrumental in the understanding of invertebrate and plant development. Here, we report the results from the first application of such a large-scale genetic screening to vertebrate development.
Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells at an average specific locus rate of one in 651 mutagenized genomes. Mutations were transmitted to the F1 generation, and 2205 F2 families were raised. F3 embryos from sibling crosses within the F2 families were screened for develop-mental abnormalities. A total of 2337 mutagenized genomes were analyzed, and 2383 mutations resulting in abnormal embryonic and early larval phenotypes were identified. The phenotypes of 695 mutants indicated involvement of the identified loci in specific aspects of embryogenesis. These mutations were maintained for further characterization and were classified into categories according to their phenotypes. The analyses and genetic complementation of mutations from several categories are reported in separate manuscripts. Mutations affecting pig-mentation, motility, muscle and body shape have not been extensively analyzed and are listed here. A total of 331 mutations were tested for allelism within their respective categories. This defined 220 genetic loci with on average 1.5 alleles per locus. For about two-thirds of all loci only one allele was isolated. Therefore it is not possible to give a reliable estimate on the degree of saturation reached in our screen; however, the number of genes that can mutate to visible embryonic and early larval phenotypes in zebrafish is expected to be several-fold larger than the one for which we have observed mutant alleles during the screen. This screen demonstrates that mutations affecting a variety of developmental processes can be efficiently recovered from zebrafish.
The mouse yolk sac has been shown to contain in-vivo colony forming cells capable of producing granulocytic, megakaryocytic and erythroid spleen colonies; in-vitro colony forming cells producing granulocytic and mononuclear-macrophage colonies in agar; and cells capable of repopuiating the lymphoid and myeloid tissue of lethally irradiated hosts. Similar haemopoietic precursor cells were also demonstrated in the blood at the time of initiation of the circulation and in the early embryonic liver.
Organ cultures of 7 day embryos with intact yolk sacs, and embryos or yolk sacs after separation have shown the autonomous nature of the development of haemopoiesis in the yolk sac and the dependence of intra-embryonic haemopoiesis, particularly in embryonic liver, on colonization by yolk sac haemopoietic cells.
Both in-vivo and in-vitro colony forming cells have been involved in the first migration stream, between yolk sac and embryonic liver, and evidence has been presented for the role of local environmental factors in controlling the differentiation of these cell types.
These results support the view that development of haemopoietic organs in both embryo and adult is dependent on colonization by circulating cells and that these circulating stem cells originate initially in the yolk sac. This indicates that the yolk sac is the only site of genuine de novo formation of haemopoietic stem cells.
A breakpoint cluster region (T-ALLbcr) has been previously described on 11p13 for T-ALL carrying t(11;14)(p13;q11). One further T-ALL breakpoint is described bringing to 5 out of 6 such translocations which are found to break within a maximum of 6.7 kb on chromosome 11p13. Studies of somatic cell hybrids derived from t(11;14)(p13;q11) T-ALL placed the T-ALLbcr between the genes for catalase (CAT) and the beta-subunit of follicle stimulating hormone (FSHB). This suggested a link between the T-ALLbcr and the Wilms' tumour predisposition locus (WT) since constitutional 11p13 deletions predispose to Wilms' tumour. Utilising somatic cell hybrids from patients with Wilms' tumours and aniridia, we show that while the T-ALLbcr maps distal to the catalase gene at 11p13, it maps outside the shortest region of overlap of a series of 11p13 deletions associated with Wilms'-Aniridia. The data suggest the order of genes at 11p13 to be: centromere-CAT-T-ALLbcr-WT-aniridia-FSHB-telomere. Therefore, the T-ALLbcr must lie very close to but may be distinct from the Wilms' predisposition locus at 11p13.
The E26 avian leukemia virus encodes a transcriptional activator-type oncoprotein consisting of Gag, Myb, and Ets domains, and transforms early erythroid cells as well as myeloblasts. Surprisingly, we have found that "early erythroid" transformants obtained in culture are multipotent, since they can be induced to differentiate into myeloblasts and eosinophils after superinfection with retroviruses containing kinase-type or ras oncogenes. In addition, TPA is an efficient inducer that generates predominantly eosinophils at low concentrations and myeloblasts at high concentrations. The determination process involves the complete extinction of erythroid/thrombocytic markers and the subsequent activation of myelomonocytic/eosinophilic properties, including the acquisition of specific growth factor requirements. "Erythroleukemic" cells from virus-infected animals were likewise found to be multipotent, making this a unique system to study the genesis of stem cell leukemias and the molecular basis of lineage commitment during hematopoiesis.
Erythropoietin, a glycoprotein produced by the kidneys in response to anemia and hypoxia, is a major growth factor for cells of the erythroid lineage. Erythropoietin interacts with high-affinity cell surface receptors (EpoR) present on developing progenitors and is required for their survival. Previously we characterized the gene for EpoR and demonstrated that its promoter acts in a cell-specific manner. Here we show that the hematopoietic-specific transcription factor GATA-1 is necessary, and indeed is sufficient as the sole cell-restricted regulator, for activation of the EpoR promoter in fibroblast transfection assays. Hence, GATA-1, which participates in transcriptional control of the majority of erythroid-expressed genes, also acts on the promoter of an essential lineage-restricted receptor (EpoR). This central contribution of GATA-1 to EpoR promoter function provides a mechanism whereby a cell-restricted regulator may ensure the viability and subsequent maturation of progenitor cells during hematopoietic differentiation.
The mature cells in the haemopoietic system arise as the result of the extensive developmental and proliferative capacity of pluripotential stem cells. In order to understand the molecular basis for these developmental processes, it will be necessary to identify and characterize the cellular genes that control early steps in haemopoiesis. Mutations at the mouse W locus on chromosome 5 lead to pleiotropic developmental defects, including sterility, coat colour abnormalities, severe macrocytic anaemia and mast cell deficiency. The defects in all these lineages are cell autonomous and intrinsic, suggesting that the W locus encodes a gene product required directly for cellular differentiation. In an attempt to understand this classical mouse developmental mutation, we have demonstrated that the c-kit proto-oncogene, which encodes a transmembrane receptor tyrosine kinase, is very closely linked to W. Several further observations are consistent with the idea that W and c-kit are allelic: first, c-kit is expressed in those cell populations affected by W mutations; second, the expression of c-kit transcripts can be affected by mutations at the W locus; third, the tyrosine kinase activity associated with the protein encoded by c-kit is functionally impaired in mast cells derived from mutant W/Wv mice; and fourth, rearrangements within the c-kit gene have been reported in two W mutant alleles. These observations suggest that the dominant phenotype associated with W mutations results from loss-of-function alterations that affect the receptor tyrosine kinase encoded by c-kit. The demonstration that the W locus encodes a transmembrane growth factor receptor provides a molecular basis for understanding the intrinsic haemopoietic defect in W mutant mice and the role that this cellular proto-oncogene plays in haemopoiesis and other developmental processes.
The t(8;21)(q22;q22) translocation is a non-random chromosomal abnormality frequently found in patients with acute myeloid leukemia (AML) with maturation (M2 subtype). We report here the cloning of a gene, named AML1, on chromosome 21 that was found to be rearranged in the leukemic cell DNAs from t(8;21) AML patients. The breakpoints in 16 out of 21 patients were clustered within a limited region of AML1, and detailed analysis in 3 patients revealed that the breakpoints occurred in the same intron of the gene. Sequencing of cDNA clones identified a long open reading frame encoding a 250-amino acid protein. Northern blot analysis detected four constant mRNA species in t(8;21) leukemic and normal cells; the largest species was more abundant in the leukemic cells than in normal cells. In addition, two mRNA species limited to the leukemic cells were found. These findings indicate that the AML1 gene may be involved in neoplastic transformation of AML with the t(8;21) translocation.
When embryonic stem cells are cultured directly in semisolid media (methyl cellulose), they proliferate and differentiate to generate colonies known as embryoid bodies (EBs). These EBs consist of differentiated cells from a number of lineages including those of the hematopoietic system. Following 10 days of culture in the presence of 10% fetal calf serum, more than 40% of all EBs from three different ES cell lines, CCEG2, D3 and SQ1.2S8 contained visible erythropoietic cells (i.e. red with hemoglobin). Beta H1 (z globin) mRNA is detectable in EBs within 5 days of differentiation, whilst beta(maj)-globin RNA appears by day 6. In the presence of erythropoietin (Epo), the frequency of EBs with erythropoietic activity increases to greater than 60%; Epo also prolongs this erythropoietic activity. Interleukin-3 (IL-3) does not significantly increase the frequency of EBs that contain erythroid cells, but increases slightly the number of erythropoietic cells associated with them. In the presence of IL-3, in addition to cells of the erythroid lineage, macrophages, mast cells and in some instances neutrophils are found within differentiating EBs. The development of macrophages is significantly enhanced by the addition of IL-3 alone or in combination with IL-1 and M-CSF or GM-CSF. When well-differentiated EBs are allowed to attach onto tissue-culture plates and grown in the presence of IL-3, a long-term output of cells from the mast cell lineage is observed.(ABSTRACT TRUNCATED AT 250 WORDS)
We have cloned 70 kb of DNA from chromosome 11p13 at the site of a recurrent translocation in T-cell leukaemia (T-ALL): t(11;14)(p13;q11). The translocation involves the TCR-delta gene on 14q11 and a new site on 11p13. Two new and 10 previously identified translocations all mapped within 25 kb on 11p13, the 11p13 T-cell translocation cluster (11p13 ttc). A search for expressed sequences surrounding the breakpoint cluster region on 11p13 identified a gene telomeric of all breakpoints which is overexpressed in three T-ALL samples with a t(11;14). The gene T-cell translocation gene (TTG-2) encodes a small cysteine-rich protein. Forty-eight per cent of the amino acids are identical with another translocation-deregulated gene, TTG-1 (T-cell translocation gene 1 or rhombotin) in 11p15. There are two copies of a cysteine-rich motif in both proteins. Two tandem copies of the same cysteine-rich motif are also present in the recently described lin-11, isl-1 and mec-3 gene products, and one motif is found in the CRIP protein. Therefore the proteins encoded by these two translocation-deregulated genes belong to this new class of cysteine-rich proteins with the 'LIM' motif, which are important in normal development.
The zinc-finger transcription factor GATA-1 (previously known as GF-1, NF-E1 or Eryf 1 binds to GATA consensus elements in regulatory regions of the alpha- and beta-globin gene clusters and other erythroid cell-specific genes. Analysis of the effects of mutations in GATA-binding sites in cell culture and in binding assays in vitro, as well as transactivation studies with GATA-1 expression vectors in heterologous cells, have provided indirect evidence that this factor is involved in the activation of globin and other genes during erythroid cell maturation. GATA-1 is also expressed in megakaryocytes and mast cells, but not in other blood cell lineages or in non-haemopoietic cells. To investigate the role of this factor in haematopoiesis in vivo, we disrupted the X-linked GATA-1 gene by homologous recombination in a male (XY) murine embryonic stem cell line and tested the GATA-1-deficient cells for their ability to contribute to different tissues in chimaeric mice. The mutant embryonic stem cells contributed to all non-haemopoietic tissues tested and to a white blood cell fraction, but failed to give rise to mature red blood cells. This demonstrates that GATA-1 is required for the normal differentiation of erythroid cells, and that other GATA-binding proteins cannot compensate for its absence.
We show that expression in fibroblasts of a single cDNA, encoding the erythroid DNA-binding protein Eryf1 (GF-1, NF-E1), very
efficiently activates transcription of a chicken alpha-globin promoter, trans-Activation in these cells occurred when Eryf1
bound to a single site within a minimal globin promoter. In contrast, efficient activation in erythroid cells required multiple
Eryf1 binding sites. Our results indicate that mechanisms exist that are capable of modulating the trans-acting capabilities
of Eryf1 in a cell-specific manner, without affecting DNA binding. The response of the minimal globin promoter to Eryf1 in
fibroblasts was at least as great as for optimal constructions in erythroid cells. Therefore, the assay provides a very simple
and sensitive system with which to study gene activation by a tissue-specific factor.
The helix-loop-helix genes LYL, SCL and E2A are associated with chromosome translocations found in human lymphoid leukemias. To establish their hematopoietic expression patterns, we have isolated murine LYL and SCL cDNA clones and investigated the expression of all three genes by Northern blot analysis of 58 murine hemopoietic cell lines and tissues. The nucleotide sequences of LYL cDNA clones revealed alternative 5' untranslated sequences and differential splicing within the 5' portion of the coding region that may produce a LYL polypeptide lacking an N-terminal segment. The LYL gene was expressed in most myeloid, erythroid and B lymphocyte cell lines and displayed two alternative size classes of transcripts, the smaller size class (1.5-1.8 kb) being typical of the erythroid lineage and the larger class (2.0-2.3 kb) of the B cell lineage. These two size classes were found to differ in the 5' untranslated region. Thus, expression of the LYL gene appears to be differentially regulated in different hemopoietic cell types. In contrast, the E2A gene was expressed throughout the hemopoietic compartment as a single dominant transcript (3.5 kb). SCL expression was restricted to erythroid, mast and early myeloid cell lines, and the level of SCL transcripts (3.0 and 4.7 kb species) increased markedly during DMSO-induced differentiation of erythro-leukemia cells. Hence the SCL gene product may be an important regulatory factor for the erythroid lineage. The low or undetectable expression of both SCL and LYL in most T lymphoid cell sources is consistent with the view that the translocations of these genes in human T cell leukemias alter their normal regulation and may thereby contribute to neoplasia.
The SCL gene encodes a member of the 'helix-loop-helix' family of DNA binding regulatory proteins. It is transcriptionally activated in some cases of T-cell acute lymphoblastic leukaemia by a reciprocal translocation involving the T cell receptor delta locus. In order to gain insight into the normal functions of SCL we have studied SCL mRNA levels in human and murine haemopoietic cell lines and normal tissues. We have observed high levels of SCL mRNA in all human and murine erythroid cell lines examined. Foetal and adult normal haemopoietic cell populations rich in erythroid precursors also expressed high levels of SCL mRNA. Our results suggest a previously unexpected role for SCL in the regulation of erythropoiesis.
Almost 30% of patients with T-cell acute lymphoblastic leukemia (T-ALL) bear structural alterations of tal-1, a presumptive
proto-oncogene that encodes sequences homologous to the helix-loop-helix (HLH) DNA-binding and dimerization domain. Analysis
of the tal-1 gene product reveals that its HLH domain mediates protein-protein interactions with either of the ubiquitously
expressed HLH proteins E47 and E12. The resultant tal-1/E47 and tal-1/E12 heterodimers specifically recognize the E-box DNA
sequence motif found in eucaryotic transcriptional enhancers. Hence, the tal-1 protein shares biochemical properties with
other tissue-specific HLH proteins that control cell type determination during myogenesis (e.g., MyoD1) and neurogenesis (e.g.,
achaete-scute). The data suggest that HLH heterodimers involving tal-1 may function in vivo as transcriptional regulatory
factors that influence cell type determination during hematopoietic development.
A powerful enhancer has been mapped to an 18-bp DNA segment located 11 kb 5' to the human epsilon-globin gene within the dominant control or locus-activating region. This enhancer is inducible in K562 human erythroleukemia cells, increasing linked gamma-globin promoter/luciferase gene expression to 170-fold over an enhancerless construct. The enhancer consists of tandem AP-1-binding sites, phased 10 bp apart, which are both required for full activity. DNA-protein binding assays with nuclear extracts from induced cells demonstrate a high molecular weight complex on the enhancer. The formation of this complex also requires both AP-1 sites and correlates with maximal enhancer activity. Induction of the enhancer may have a role in the increase in globin gene transcription that characterizes erythroid maturation. Enhancer activity appears to be mediated by the binding of a complex of proteins from the jun and fos families to tandem AP-1 consensus sequences.
Congenital dyserythropoietic anaemia Type II or HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum lysis test) is a rare genetic anaemia in humans, inherited in an autosomally recessive mode. Biochemical analyses of HEMPAS erythrocyte membranes suggested strongly that HEMPAS is caused by defective glycosylation of erythrocyte membrane glycoproteins. Most recently a HEMPAS case has been identified as being defective in the gene encoding Golgi alpha-mannosidase II by using cDNA probe of alpha-mannosidase II. At present, it is not clear whether HEMPAS is a genetically heterogenous collection of glycosylation deficiencies, as some HEMPAS cases showed a low level of N-acetylglucosaminyltransferase II. Abnormal glycosylation of serum glycoproteins and association of liver cirrhosis in HEMPAS patients indicate that HEMPAS disease is not restricted to erythroid cells. On the other hand, normal development of HEMPAS patients during embryonic stage strongly suggests the possibilities of fetal type isozyme in place of defective glycosylation enzyme.
Suggestions that the field of hemoglobin regulation and erythroid cell molecular biology was undergoing a tortuous and slow death, awash in the scientific community several years ago, were dispelled by the findings presented at the Seventh Conference on Hemoglobin Switching. After a phase in which neither the cis-elements nor trans-factors important for globin and erythroid gene expression were evident, recent progress has been rapid. Once again, studies in this area are providing fundamental insights into eukaryotic biology. The long-distance influence of LCR elements on chromatin structure and gene expression is remarkable and likely to be encountered in the analysis of other developmentally regulated, multigene loci. How LCR elements influence chromatin structure and maintain an open configuration is a problem at the core of gene regulation. We can be optimistic that further dissection of LCRs will delineate DNA sequences critical for these effects and associated proteins. The interaction of LCRs with individual genes must depend on specific protein-protein interactions, most likely involving a small, but elite, group of regulators. At least one critical transcriptional regulator of erythroid-expressed genes, GATA-1, is firmly established. Others are being pursued. The mechanisms by which they collaborate with each other should provide the missing pieces to the puzzle of cell-specific gene expression in the erythroid lineage. As the phenomenology of Hb switching is mimicked in transgenic mice, the elements mediating competitive and non-competitive (or autonomous) modes of regulation will be systematically delineated. Whether knowledge of the cis- and trans- components involved in switching will lead to the development of therapeutic approaches aimed at altering their complex interactions is uncertain. Fortunately, recent progress in hematopoietic stem cell biology once again raises hopes that gene transfer strategies for management of hemoglobin disorders may be more than a distant, impractical goal.
NF-E1, a DNA-binding protein that recognizes the general consensus motif WGATAR, is the first tissue-specific factor to be identified in erythroid cells. Using a probe from the murine GF-1 (NF-E1) cDNA clone, we isolated three homologous chicken cDNAs: One of these corresponds to an mRNA (NF-E1a) that is abundantly and exclusively expressed in erythroid cells; a second mRNA (NF-E1b) is also expressed in all developmental stages of erythroid cells but is additionally found in a limited subset of other chicken tissues; mRNA representative of a third gene (NF-E1c) is expressed only in definitive (adult) erythrocytes within the red cell lineage but is also abundantly expressed in T lymphocytes and brain. All NF-E1 proteins are highly conserved within the DNA-binding domain and bind to the consensus motif with similar affinities in vitro; they are also all stimulatory trans-acting factors in vivo. The factors differ quantitatively in their ability to trans-activate reporter genes in which the number and position of cognate binding sites is varied relative to the transcriptional initiation site. These data suggest that the NF-E1 consensus motif directs a broader and more complicated array of developmental transcriptional regulatory processes than has been assumed and that NF-E1c may play a unique regulatory role in the developing chicken brain and in T lymphocytes.
In zebrafish, as in Xenopus, the well-orchestrated cell movements of gastrulation can be dissected into several components, including epiboly, involution, convergence and extension. Embryos homozygous for the recessive lethal mutation spt-1(b104) or 'spadetail' have a complex set of defects in the trunk of the embryo that may arise secondarily after loss of one of these movements, convergence, from those precursors that would normally have given rise to trunk somitic mesoderm. We have now tested this hypothesis by transplanting cells between wild-type and mutant embryos, to identify the cells that spt-1 affects directly. Our results show that the mutation autonomously affects only those mesodermal precursors located along the lateral margin of the early gastrula blastoderm. Other mesodermal cells and all ectodermal precursors seem not to require function of the wild-type gene. Our findings reveal an unexpectedly delicate genetic control of vertebrate gastrulation.
We have analyzed t(1;14)(p32;q11) chromosome translocations from two patients with T cell acute lymphocytic leukemia. The chromosome 1 breakpoints of these patients lie within a kilobasepair of each other, and thus define a genetic locus (designated tal) involved in T cell oncogenesis. Moreover, we have identified sequences within tal that potentially encode an amphipathic helix-loop-helix motif, a DNA-binding domain found in a variety of proteins that control cell growth and differentiation. The homology domain of tal is especially related to that of lyl-1, a gene on chromosome 19 that has also been implicated in T cell oncogenesis. Hence, tal and lyl-1 encode a distinct family of helix-loop-helix proteins involved in the malignant development of lymphocytes.
Erythroid-specific genes contain binding sites for NF-E1 (also called GF-1 and Eryf-1; refs 1-3 respectively), the principal DNA-binding protein of the erythrocytic lineage. NF-E1 expression seems to be restricted to the erythrocytic lineage. A closely related (if not identical) protein is found in both a human megakaryocytic cell line and purified human megakaryocytes; it binds to promoter regions of two megakaryocytic-specific genes. The binding sites and partial proteolysis profile of this protein are indistinguishable from those of the erythroid protein; also, NF-E1 messenger RNA is the same size in both the megakaryocytic and erythroid cell lines. Furthermore, point mutations that abolish binding of NF-E1 result in a 70% decrease in the transcriptional activity of a megakaryocytic-specific promoter. We also find that NF-E2, another trans-acting factor of the erythrocytic lineage, is present in megakaryocytes. Transcriptional effects in both lineages might then be mediated in part by the same specific trans-acting factors. Our data strengthen the idea of a close association between the erythrocytic and the megakaryocytic lineages and could also explain the expression of markers specific to the erythrocytic and megakaryocytic lineages in most erythroblastic and megakaryoblastic permanent cell lines.
The nuclear factor GF-1 (also known as NF-E1, Eryf-1; refs 1-3 respectively) is important in regulation of the transcription of globin and other genes that are specifically expressed in erythroid cells. We have previously shown that GF-1 of both mouse and human origin is a 413-amino-acid polypeptide with two novel zinc-finger domains whose expression is restricted to erythroid cells. Using in situ hybridization of mouse bone marrow cells and northern blot analysis of purified cell populations and permanent cell lines, we show here that GF-1 is expressed in two other hematopoietic lineages, megakaryocytes and bone marrow-derived mast cells. Our findings are consistent with results from hematopoietic progenitor culture which suggest a relationship between erythroid, megakaryocytic and mast cell lineages, and imply that GF-1 is expressed in committed multipotential cells and their progeny. Hence, the mere presence of this transcription factor is unlikely to be sufficient to programme differentiation of a single haematopoietic lineage. GF-1 may regulate the transcription of not only erythroid genes, but also many genes characteristic of megakaryocytes and mast cells, or genes shared among these lineages.
The mechanism by which erythropoietin controls mammalian erythrocyte production is unknown. Labeling experiments in vitro
with [3H]thymidine demonstrated DNA cleavage in erythroid progenitor cells that was accompanied by DNA repair and synthesis.
Erythropoietin reduced DNA cleavage by a factor of 2.6. In the absence of erythropoietin, erythroid progenitor cells accumulated
DNA cleavage fragments characteristic of those found in programmed cell death (apoptosis) by 2 to 4 hours and began dying
by 16 hours. In the presence of erythropoietin, the progenitor cells survived and differentiated into reticulocytes. Thus,
apoptosis is a major component of normal erythropoiesis, and erythropoietin controls erythrocyte production by retarding DNA
breakdown and preventing apoptosis in erythroid progenitor cells.
The nuclear protein encoded by the proto-oncogene c-myb has been hypothesized to play an important role in the process of
hematopoiesis, but direct proof of this function has been lacking. To address this issue, normal human bone marrow mononuclear
cells were exposed to c-myb sense and antisense synthetic oligodeoxynucleotides, and the effects on hematopoietic colony formation
and maturation were examined. Exposure of these cells to c-myb antisense, oligodeoxynucleotides resulted in a decrease in
both colony size and number, without apparent effect on the maturation of residual colony cells. Exposure to c-myb sense,
or irrelevant antisense, oligonucleotides had no such effect. These results show that (i) c-myb plays a critical role in regulating
normal human hematopoiesis and (ii) the combined use of antisense oligodeoxynucleotides and hematopoietic cell culture techniques
will provide a powerful tool for studying the role of proteins encoded by proto-oncogenes, or other specific genes, in normal
human hematopoiesis.
Relative levels of the nuclear oncoproteins c-myb, c-myc, and c-fos were determined in selected subpopulations of normal human bone marrow (BM) cells using a flow cytometric assay which simultaneously detects a cell-surface antigen (as a marker of lineage and stage of maturation) and levels of an intracellular protein. At least two monoclonal antibodies directed against each oncoprotein and specific peptide inhibition controls were used for these determinations. Hematopoietic progenitor cells (CD34+) express the highest levels of c-myb and c-myc, whereas c-fos levels in CD34+ progenitor cells are similar to c-fos levels in mature monocytes and granulocytes. Granulocytes are the only hematopoietic cells examined which do not express detectable levels of c-myb and c-myc. The levels of these oncoproteins in these normal, unstimulated BM cell populations were more closely linked to lineage and maturation stage than to the proliferative status of the given population, as determined by either DNA staining or expression of the cell-cycle specific nuclear protein, Ki67. This flow cytometric assay helps in interpreting the significance of oncoprotein levels in leukemia cells by allowing direct comparisons of a leukemia with the phenotypically similar "normal counterpart control" cell population in normal BM.
Two independent cDNA clones encoding the erythropoietin receptor (EPO-R) were isolated from a pXM expression library made from uninduced murine erythroleukemia (MEL) cells. The clones were identified by screening COS cell transfectants for binding and uptake of radioiodinated recombinant human erythropoietin. As inferred from the cDNA sequence, the murine erythropoietin receptor is a 507 amino acid polypeptide with a single membrane-spanning domain. It shows no similarities to known proteins or nucleic acid sequences in the data bases. Although the MEL cell EPO-R has a single affinity with a dissociation constant of approximately 240 pM, the EPO-R cDNA, expressed in COS cells, generates both a high-affinity (30 pM) and a low-affinity (210 pM) receptor.
Quail-chick intracoelomic grafts of organ rudiments were used to study the origin of endothelia and haemopoietic cells during avian organogenesis in conjunction with the monoclonal antibody QH1 which recognizes the quail haemangioblastic lineage. Results differed according to the germ-layer constitution of the grafted rudiments. In the case of the limb buds, endothelial cells from the host invaded the graft through an angiogenic process. Haemopoietic progenitors from the host also colonized the grafted bone marrow. In contrast, rudiments of internal organs provided their own contingent of endothelial precursors, a process termed vasculogenesis. Nevertheless, haemopoietic cells in these organs were all derived from the host. In the lung, this extrinsic cell population appeared regularly scattered around the parabronchi and had a macrophage-like phenotype. In the pancreas, the granulocytes which differentiate as dense aggregates located in the wall of the largest vessels were extrinsic. Similarly in the spleen, a mesodermal primordium that develops in close association with the pancreatic endoderm, endothelial cells were intrinsic and haemopoietic cells host-derived. This study demonstrates that, in ontogeny, vascularization obeys different rules depending on which germ layer the mesoderm is associated with: in mesodermal/ectodermal rudiments angiogenesis is the rule; in mesodermal/endodermal rudiments, vasculogenesis occurs. However, in these internal organs undergoing vasculogenesis, endothelial and haemopoietic cells have separate origins. We put forward the hypothesis that the endoderm induces the emergence of endothelial cells in the associated mesoderm. Formation of blood stem cells may also involve interactions between endoderm and mesoderm, but in this case the responding capacity of the mesoderm appears restricted to the paraaortic region.
Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.
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.
The erythroid-specific transcription factor Eryf1 binds to DNA sites within regulatory regions of every member of both the alpha- and beta-globin families in chicken. The distribution of these sites suggests that Eryf1 may serve as a general "switch" factor for erythroid development. We have cloned the cDNA for Eryf1 and show that the corresponding mRNA is present in all erythroid lineages, but is absent from non-erythroid cells. We demonstrate that the cDNA encodes the specific Eryf1 binding activity found in erythrocytes. Eryf1 is a basic 38 kd protein containing a pair of highly similar "fingers" with the motif Cys-x-x-Cys-x17-Cys-x-x-Cys. The amino acid sequences of these regions bear no resemblance to those found in other regulatory proteins with a similar arrangement of cysteine residues. Our evidence suggests, furthermore, that transition metal ions are unusually tightly bound, or may not be necessary for the sequence-specific DNA binding of Eryf1.
A full-length human c-myb cDNA clone has been isolated from a CCRF-CEM leukemia cell cDNA library. The plasmid vector contains
simian virus 40-derived promotor, splice, and polyadenylation sequences as well as a transcription unit for a dihydrofolate
reductase cDNA. We have introduced this construct into Friend erythroleukemia (F-MEL) cells and have isolated a number of
clones which contain intact and transcriptionally active human c-myb sequences. F-MEL clones expressing the highest levels
of the human c-myb mRNA differentiate poorly in response to dimethyl sulfoxide. Two clones which initially expressed low levels
of human c-myb transcripts and which differentiated normally were subsequently inhibited in their ability to differentiate
when grown in successively higher concentrations of methotrexate, due to amplification and enhanced expression of plasmid
sequences. The inhibitory effect on F-MEL differentiation appeared to be independent of the early decline in c-myc transcripts
which were normally regulated in all cases examined. Our results indicate that constitutive expression of a nontruncated human
c-myb cDNA can exert profound effects on erythroid differentiation and argue for a causal role of c-myb in the F-MEL differentiation
process.
Erythropoietin, GM-colony-stimulating factor and G-colony-stimulating factor are the first recombinant haemopoietic growth factors to reach clinical use. There are a number of additional haemopoietic regulators that have now been cloned and are being mass-produced with a view to clinical use. The next decade should witness exciting advances in the clinical treatment of haematological diseases and infections that will be comparable with those that were seen last with the introduction of effective treatments for pernicious anaemia.
We have identified a protein present only in erythroid cells that binds to two adjacent sites within an enhancer region of the chicken beta-globin locus. Mutation of the sites, so that binding by the factor can no longer be detected in vitro, leads to a loss of enhancing ability, assayed by transient expression in primary erythrocytes. Binding sites for the erythroid-specific factor (Eryf1) are found within regulatory regions for all chicken globin genes. A strong Eryf1 binding site is also present within the enhancer of at least one human globin gene, and proteins from human erythroid cells (but not HeLa cells) bind to both the chicken and the human sites.
We have constructed a "minilocus" that contains the 5' and 3' flanking regions of the human beta-globin locus and the beta-globin gene. These regions are characterized by erythroid-specific DNAase I-superhypersensitive sites and are normally located approximately 50 kb 5' and 20 kb 3' of the beta-globin gene. This minilocus is expressed tissue-specifically in transgenic mice at a level directly related to its copy number yet independent of its position of integration in the genome. Moreover, the expression per gene copy is the same in each mouse and as high as that of the endogenous mouse beta-globin gene. These results indicate that the DNA regions flanking the human beta-globin locus contain dominant regulatory sequences that specify position-independent expression and normally activate the complete human multigene beta-globin locus.