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作者：Anna Rita Migliaccioa, Sjaak Philipsenb作者单位：aIstituto Superiore di Sanit, Rome, Italy;bErasmus University Medical Center, Department of Cell Biology, Rotterdam, The Netherlands / B! J+ u& q$ J7 y


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      【摘要】" Q$ @" N* u+ K! `+ @  x! p5 f
   Red blood cells (RBCs) mediate oxygen transport throughout the body, a function that is essential for life. RBCs are continuously produced via a process called erythropoiesis. Anemias (insufficient numbers of functional RBCs), caused by failure of erythropoiesis, are a major cause of disease worldwide. Hereditary anemias constitute the most common human genetic disorders; they have no effective cure yet. The European research training network Eurythron follows a multidisciplinary approach to clarify the important molecular mechanisms in normal and pathological erythropoiesis, with a view to develop novel therapies to cure the anemias. The aim is to generate a comprehensive molecular description of mechanisms governing specification of hematopoietic stem cells in embryogenesis, lineage commitment, differentiation, and postmitotic maturation of RBCs. We report on the Eurythron meeting in Rome, in which novel approaches in stem cell and erythroid cell biology, including in vitro expansion of primary cells, biochemistry of receptor/signal transduction complexes and transcription factors, and (epi)genetics, were discussed. + |0 v8 P/ \# T0 e4 {. i) P$ r
      【关键词】 Hematopoietic stem cells Commitment Hematopoiesis Erythropoiesis Networks% j) c& R) F" f3 J
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The process of hematopoiesis continues to play a central role in establishing the general principles underlying how adult stem cells arise during embryogenesis and the decisions they make in forming fully differentiated mature cells. Historically, the hematopoietic model has prevailed because progenitors are accessible and can be analyzed at different stages of differentiation using well-established high-speed cell sorting and imaging methods. Purified cells can then be characterized using a variety of techniques to determine their transcriptional profiles, epigenetic programs, and responses to alterations in cell signaling. Importantly, many of these processes are well conserved throughout evolution, for instance, in zebrafish, Xenopus, mouse, and human, allowing one to extend and extrapolate results from one species to another. Eva Krpelanova and Sjaak Philipsen (Erasmus University Medical Center, Rotterdam, The Netherlands) presented the use of the roundworm Caenorhabditis elegans as a tool for functional studies of transcription factor families. Although C. elegans does not have a system equivalent to the hematopoietic system of vertebrates, it does contain homologues representing the many and often large vertebrate transcription factor families with hematopoietic functions. C. elegans is an attractive model system for the analysis of basic biological problems, including stem cell biology. Unique to this organism is the fact that the origin and developmental fate of every cell are known. This fact, in combination with the complete genome sequence, comprehensive and accessible collections of mutants, and a full arsenal of molecular and genetic tools, makes it feasible to unravel the role of entire gene families in ontogeny and lineage determination of every cell of the organism. Although more time consuming, the use of homologous recombination, transgenic approaches, viral transduction systems, microinjection, single chain antibodies, RNA interference, and morpholinos has enabled comprehensive experimental approaches to address the key issues in vertebrate organisms. The rich and still growing resource of natural and experimentally induced mutations has directed relevant scientific questions and also provided a clinical perspective.( x6 z  c+ t2 P, L, ~2 z$ V: X

Hematopoietic Stem Cells/ D2 ?& G, ^: [

How are hematopoietic stem cells generated during development and how are they maintained in adult life? How do they proliferate and control stem cell numbers? How do they choose among self-renewal, lineage commitment, and differentiation? First, it has been important to establish the precise cellular origins of hematopoietic stem cells (HSCs) from mesoderm. Mesoderm induction is the first step leading to the specification of embryonic and adult hematopoietic cells. Lineage labeling in Xenopus has shown that these two blood populations arise from different parts of the embryo giving rise to transient embryonic (primitive) hematopoiesis and permanent (definitive) hematopoiesis . This raises the exciting possibility that the human placenta is also an abundant source of HSCs, which could be used for therapeutic applications. It is not trivial to extend these observations from the mouse to the human, since there are major differences in the tissue architecture of the human and mouse placenta, and it is unknown at which developmental stage HSCs might be present in the human placenta. It also remains to be determined whether placental HSCs can be expanded in vitro and whether HSCs can be generated in situ in the placenta.

Lineage Determination: y, m; B' {* r- s) p

Many approaches exist to unravel the process by which a HSC becomes committed to the erythroid lineage. Since the phenotype of a given cell ultimately reflects the genes it currently expresses or has expressed in the past, research has focused on (a) analyzing global gene expression profiles, both at the transcriptional and the translational levels; (b) the expression of individual genes that are already known to play an important role in hematopoiesis (e.g., the transcription factors Gata1, Gata2, Runx1, and Tal1); and (c) emerging signaling pathways (e.g., those activated by integrins; Wnts/ß-catenin, Notch, and receptor tyrosine kinases), which may crucially regulate the activity of transcriptional/epigenetic regulators, and thus influence decisions between renewal and commitment. Tariq Enver (The Weatherall Institute of Molecular Medicine) discussed the development of mathematical models to describe how cells make transitions between stable states. It is known that the transcription factors Gata1 and Pu.1 have a cross-antagonistic relationship: they are involved in a positive autoregulatory feedback loop but repress each other's activity .
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Erythroid-Restricted Progenitors

Following specification of the bipotent megakaryocyte-erythrocyte progenitor from the multipotent common myeloid progenitor, the earliest recognizable cell committed to the erythroid lineage is the erythroid burst-forming unit (BFU-E). This cell is defined by its ability to form large erythroid colonies in vitro. Under the influence of a variety of cytokines and growth factors, the corresponding progenitors in vivo become further committed to the erythroid lineage, proliferate, and differentiate to mature red cells. There are a number of protocols that enable both human and mouse erythroid precursors to be purified and subdivided into different stages of maturation, and unlike earlier precursors, such cells can be obtained in larger numbers. In addition, protocols have recently been developed for long-term expansion of mouse and human BFU-E-like progenitors . The combined use of molecular biology and advanced confocal microscopy will enable an in vivo analysis of the protein domains involved in the dynamics of nuclear localization.' C0 G  P& w/ s% G9 _7 ?
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Transcription Factor Complexes/ z  A) v4 p& [7 \
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Some key transcription factors involved in erythropoiesis are already known (e.g., Gata1, Gata2, EKLF, NF-E2, Nrf1¨C3, and Tal1). Newly developed procedures for in vivo tagging of these proteins enables the isolation of their interacting partners. John Strouboulis (Erasmus MC) discussed the application of in vivo biotinylation tagging of transcription factors, using a small peptide tag recognized by the Escherichia coli biotin ligase BirA. Biotinylated factors can be isolated with high efficiency and specificity in a single-step purification procedure, followed by mass spectrometry analysis to identify potential interaction partners  (N. Meier, S. Krpic, P. Rodriguez, J. Strouboulis, M. Monti, J. Krijgsveld, M. Gering, R. Patient, F. Grosveld, A. Hostert, manuscript submitted for publication). Morpholino-mediated knockdown of eto2 in zebrafish indicates that it is important for the development of the definitive hematopoietic system. Eto2 may respond to extracellular signals by translocating from the cytoplasm into the nucleus, whereas it is downregulated in terminally differentiating cells. This suggests that Eto2 plays a key role in the birth of the definitive HSCs and is required for the subsequent phases of expansion of the different lineages (N. Meier et al., manuscript submitted for publication).
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Cell Signaling
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An important aspect of understanding normal erythropoiesis is to establish the role of external signals, such as erythropoietin (Epo) and stem cell factor, in controlling proliferation and differentiation through their receptors, EpoR and cKit. EpoR is known to signal via multiple redundant pathways, which prevent premature apoptosis of the precursors and stimulate their proliferation. cKit plays a major role in controlling proliferation by increasing the Epo responsiveness of the precursors. The characterization of the scaffolding proteins of the EpoR and cKit helps to understand their responsiveness (EpoR) and specificity (cKit) in erythropoiesis. EpoR signaling is very frequently affected in the group of acquired disorders referred to as the myelodysplasias (MDSs) that represent a common, heterogeneous group of blood disorders in the elderly. The incidence of MDSs is estimated to be 14 per 105 people per year in the 65¨C85-year-old age group. A large proportion of these patients develop acute myeloid leukemia with a poor prognosis. Patrick Mayeux (Institut Cochin) presented a proteomics analysis of the EpoR signaling. His data indicate that, after ubiquitination, activated EpoR is quickly degraded by two proteolytic systems. The proteasomes remove part of the intracellular domain while the EpoR is still at the cell surface. Next, the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes offers an explanation for the short duration of intracellular signaling activated by Epo. These data have implications not only for the pathophysiological explanation of some types of MDS but also for Epo hyper-responsiveness occurring in polycythemia vera, a condition characterized by overproduction of erythrocytes. The link between cell signaling and translational control of mRNAs was discussed by the laboratory of Marieke von Lindern (Erasmus MC). Previous gene expression profiling analyses of polysome-bound mRNA had shown that translational regulation is common during erythroid differentiation . It is of interest to note that the 5'-untranslated regions of mRNAs that are subject to translational control are notably absent from the expressed sequence tag databases, likely because of their propensity to form tertiary structures that are not easily copied into cDNA. This has probably contributed to the relative underexposure of the importance of translational control in hematopoiesis.
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Terminal Differentiation of Erythroid Cells
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During the terminal stages of erythroid differentiation (proerythroblast to reticulocyte) nearly all protein synthesis is directed to the production of hemoglobin. The fully mature, enucleated red cell is a highly concentrated solution of hemoglobin surrounded by a cytoplasmic membrane. In humans, the synthesis of hemoglobin is regulated by the -globin (Tel----cen) and ß-globin (Cen--g-a--ß-tel) loci. Natural mutants of the globin genes underlie the most common inherited diseases throughout the world, the - and ß-thalassemias and sickle cell disease. The two clusters are therefore among the most intensively studied of all mammalian gene loci, but many important questions at the forefront of our attempts to understand gene regulation remain unanswered. One of these questions is the quality control of RNA transcripts, which is surveyed in the nucleus to discriminate normal from aberrant mRNAs. Splicing-defective mutants of globins cause thalassemia, even though the protein-encoding sequences are completely normal. These defective RNAs accumulate in the nucleus at the site of transcription , yet conditional deletion of Atrx in mice has no appreciable effect on -globin levels. Mice carrying the human -globin locus in lieu of their endogenous locus may finally provide an appropriate model for the study of human -globin gene expression and the pathological consequences of defective transcriptional regulation caused by impaired transcription factor function.! d% {9 g& e2 Q4 e' z

Complex Pathology of Diseases Triggered by Defective Hematopoietic Transcription Factors" L) x, B% j$ l" e

Molecular defects, either inherited or acquired, in genes encoding lineage-specific transcription factors may trigger a cascade of molecular defects only partially identifiable by gene expression profiling analysis. An example is Gata1, a transcription factor essential for proper formation of erythroid, megakaryocytic, eosinophilic, and mastocytic cells . These high levels of TGF-ß induce fibroblasts to produce collagen, thus causing marrow fibrosis. Megakaryocytes are engaged in cell-cell interactions with numerous cell types involved in the regulation of stem cell biology. It is therefore possible that pathological megakaryocyte interactions with osteoblasts, endothelial cells, or other cell types induce the abnormal stem cell trafficking and neoangiogenesis observed in the hypomorphic Gata1 mice. These data emphasize the importance of an integrated approach to the field of stem cell biology, covering the analysis of stem cell emergence, lineage determination, and terminal differentiation through the application of powerful multidisciplinary experimental techniques and model systems.

DISCLOSURES( b2 D. x% a2 I; b
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS

This work was supported by Marie Curie Research Training Network Eurythron Grant 005499.
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