细胞命运转变——谱系重编程技术研究进展

体细胞核移植和诱导多能干细胞技术表明已分化的体细胞可以转变命运。最近的研究再一次验证了成熟体细胞可以通过外源转录因子的导入,直接重编程为其他类型的体细胞或祖细胞。这种重编程技术称为谱系重编程(Lineage reprogramming)。这项技术不仅在再生医学领域具有广阔的应用前景,而且在动物生物技术中也应用广泛。它不但避免了伦理争议,还提供了便利的重编程方法,同时也为基因表达调控的研究提供了重要的手段。文章从谱系重编程的方式、谱系重编程的特点及应用前景等3个方面进行了综述,旨在对相关领域的研究人员起到借鉴作用。     

Primitive and definitive blood share a common origin in Xenopus: a comparison of lineage techniques used to construct fate maps

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

Lineage and fate in Drosophila: role of the gene tramtrack in sense organ development

 The tactile bristles of the fly comprise four cells that originate from a single precursor cell through a fixed lineage. The gene tramtrack (ttk) plays a crucial role in defining the fates of these cells. Here we analyse the normal pattern of expression of ttk, as well as the effect of ttk overexpression at different steps of the lineage. We show that ttk is never expressed in cells having a neural potential, and that in cells where ttk is expressed, there is a delay between division and the onset of expression. The ectopic expression of ttk before some stage of the cell cycle can block further cell division. Furthermore, this expression transforms neural into non-neural cells, suggesting that ttk acts as a repressor of neural fate at each step of the lineage. Our results suggest that ttk is probably not involved in setting up the mechanism that creates an asymmetry between sister cells, but rather in the implementation of that choice. Received: 10 October 1996 / Accepted: 11 February 1997     

Diacylglycerol metabolism attenuates T-cell receptor signaling and alters thymocyte differentiation

Diacylglycerol (DAG) metabolism has a critical function in Ras-regulated functions in mature T cells, but causal data linking defects in DAG-based signals with altered thymus development are missing. To study the effect of increased DAG metabolism in T-cell development, we engineered a membrane-targeted constitutive active version of DAG kinase-α (DGKα). We show that transgenic expression of constitutive active DGK leads to developmental defects in T cells, with a marked accumulation of immature CD8 thymocytes and a reduction in positive selected populations. These alterations are reflected in the periphery by a CD4/CD8 cell imbalance and general T-cell lymphopenia. The results link DAG metabolism to T-cell homeostasis, and show that correctly controlled generation and consumption of this lipid at the plasma membrane ensure T-cell passage through quality-control checkpoints during differentiation.     

Integrated pseudotime analysis of human pre-implantation embryo single-cell transcriptomes reveals the dynamics of lineage specification

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Cell lineage and gene expression in the development of polychaetes

The developmental strategies of embryos within the various taxa of polychaetes are designed to set up the fate of the cell lines. Some of these traits of pattern formation are considered to be ancestral, but we also find a number of derived developmental characteristics, some of which might be useful indicators for phylogenetic relationships. A combination of ooplasmic segregation and anisotropic cleavage rapidly establishes the fate of several larval cell lines in the polychaete embryo. The setting-up of the primary trochoblasts basically concerns the same cell line (la²–ld²) in polychaetes and even in molluscs. Such mechanisms may thus be regarded as ancestral. The determination of the mesoderm precursor occurs very late in both equally cleaving annelids and mollusks, indicating that an equal cleavage pattern may be regarded as an ancestral trait. Since both disproportionate cytoplasmic distribution (either by spindle shift or polar lobe formation) and cell cycle asynchronies appear to speed up the development of the mesoderm-forming cell line, these strategies represent derived traits. An analysis of these derived traits of early development is given and is discussed in the light of the phylogenetic relationships among the polychaetes. These data are extended by an analysis of some of the postlarval structures in polychaetes and the molecular developmenta1 circuitry involved.     

黑腹果蝇细胞谱系分析方法进展

对动物体内单个细胞的谱系进行分析有助于追踪其在发育过程中的作用,但是体内各种组织都是由很多形态、结构、功能各不相同的细胞构成的复杂系统,这种复杂性严重阻碍了对单个细胞的研究。嵌合克隆技术(Mosaic technique)和标记技术(Labeling technique)的出现为这一研究提供了强有力的手段。文章介绍了近几年来黑腹果蝇(Drosophila melanogaster)研究中常用的7种嵌合克隆标记方法,包括FRT介导的有丝分裂重组(FRT-mediated mitotic recombination)、MARCM(Mosaic analysis with a repressible cell marker)、TSG(Twin spotgenerator)、Twin-spot MARCM、Q-MARCM(Q system-based MARCM)、Coupled MARCM和G-TRACE(Gal4technique for real-time and clonal expression)技术,详述了这些技术的原理及应用,并对不同技术进行了对比。运用这些技术研究者可以从单细胞水平进行遗传学标记和操作,特别是在神经系统等复杂系统中追踪单个细胞的发育过程。果蝇中的这些技术也将为其他模式生物追踪细胞谱系提供参考。     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

Thymic medullary type epithelial cell line (MTEC1), which expressed H-2D^d and Ia^d, was derived from BALB/c mouse. MTEC1 cells were introduced by intrathymic injection into irradiated H-2^b mice reconstituted with H-2^bxd F1bone marrow cells. Two months later, the injected MTEC1 cells were found to be still present in the recipient thymus. Splenocytes from chimeric mice, inin vitro functional assays, were analyzed to investigate whether the MTEC1 cellsin vivo could induce the production of H-2^d restricted antigen-specific T cells. The H-2^d restricted VSV-antigen specific proliferating and IL-2 producing T cells as well as H-2^d restricted influenza virus specific cytotoxic T cells were found in chimeric mice injected with MTEC1 cells, and these cells were shown to be tolerant to H-2^d selfantigen. On the contrary, H-2^d restricted antigen-specific and H-2^d self-antigen tolerant T cells were not shown in control mice injected with saline. These results suggest that intrathymically injected MTEC1 cells could induce T lineage cell development and functional maturation in the intact thymus. A hypothesis of “second thymic selection” in thymic medulla has been postulated and its implication discussed. Project supported by the National Natural Science Foundation of China (Grant No. 39230320).     

Effect of mouse thymic medullary epithelial cell line on thymocyte development and functional maturation in vivo

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Embryonic lineage tracing with Procr-CreER marks balanced hematopoietic stem cell fate during entire mouse lifespan

The functional heterogeneity of hematopoietic stem cells (HSCs) has been comprehensively investigated by single-cell transplantation assay. However, the heterogeneity regarding their physiological contribution remains an open question, especially for those with life-long hematopoietic fate of rigorous self-renewing and balanced differentiation capacities. In this study, we revealed that Procr expression was detected principally in phenotypical vascular endothelium co-expressing Dll4 and CD44 in the mid-gestation mouse embryos, and could enrich all the HSCs of the embryonic day 11.5 (E11.5) aorta-gonad-mesonephros (AGM) region. We then used a temporally restricted genetic tracing strategy to irreversibly label the Procr-expressing cells at E9.5. Interestingly, most labeled mature HSCs in multiple sites (such as AGM) around E11.5 were functionally categorized as lymphomyeloid-balanced HSCs assessed by direct transplantation. Furthermore, the labeled cells contributed to an average of 7.8% of immunophenotypically defined HSCs in E14.5 fetal liver (FL) and 6.9% of leukocytes in peripheral blood (PB) during one-year follow-up. Surprisingly, in aged mice of 24 months, the embryonically tagged cells displayed constant contribution to leukocytes with no bias to myeloid or lymphoid lineages. Altogether, we demonstrated, for the first time, the existence of a subtype of physiologically long-lived balanced HSCs as hypothesized, whose precise embryonic origin and molecular identity await further characterization.     

Numb/Parkin-directed mitochondrial fitness governs cancer cell fate via metabolic regulation of histone lactylation

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Sterols are required for cell‐fate commitment and maintenance of the stomatal lineage in Arabidopsis

Asymmetric cell division is important for regulating cell proliferation and fate determination during stomatal development in plants. Although genes that control asymmetric division and cell differentiation in stomatal development have been reported, regulators controlling the process from asymmetric division to cell differentiation remain poorly understood. Here, we report a weak allele (fk–J3158) of the Arabidopsis sterol C–14 reductase gene FACKEL (FK) that shows clusters of small cells and stomata in leaf epidermis, a common phenomenon that is often seen in mutants defective in stomatal asymmetric division. Interestingly, the physical asymmetry of these divisions appeared to be intact in fk mutants, but the cell‐fate asymmetry was greatly disturbed, suggesting that the FK pathway links these two crucial events in the process of asymmetric division. Sterol profile analysis revealed that the fk–J3158 mutation blocked downstream sterol production. Further investigation indicated that cyclopropylsterol isomerase1 (cpi1), sterol 14α–demethylase (cyp51A2) and hydra1 (hyd1) mutants, corresponding to enzymes in the same branch of the sterol biosynthetic pathway, displayed defective stomatal development phenotypes, similar to those observed for fk. Fenpropimorph, an inhibitor of the FK sterol C–14 reductase in Arabidopsis, also caused these abnormal small‐cell and stomata phenotypes in wild‐type leaves. Genetic experiments demonstrated that sterol biosynthesis is required for correct stomatal patterning, probably through an additional signaling pathway that has yet to be defined. Detailed analyses of time‐lapse cell division patterns, stomatal precursor cell division markers and DNA ploidy suggest that sterols are required to properly restrict cell proliferation, asymmetric fate specification, cell‐fate commitment and maintenance in the stomatal lineage cells. These events occur after physical asymmetric division of stomatal precursor cells.     

The use of gene function to identify the rate-limiting steps controlling cell fate

The size and complexity of the genomes of mammals in general, and humans in particular, is such that it will take many years to utilise this information to produce a genuine understanding of the control of cell behaviour. Since there are tens of thousands of genes to consider, the task of identifying those which play the most significant roles, biologically and medically, is both crucial and very demanding. Here we emphasise the importance of functional approaches to answering this question, i.e. the application of techniques which use the function of the gene itself in identifying the critical rate-limiting steps in biological processes. In this review, we use the functional analysis of one of the most important of these processes, the control of survival and apoptosis, to illustrate the power of a number of functional genomic strategies.This work was presented at the first Cancer Immunology and Immunotherapy Summer School, 8–13 September 2003, Ionian Village, Bartholomeio, Peloponnese, Greece.