Intermediate filament genes as differentiation markers in the leech <Emphasis Type="Italic">Helobdella</Emphasis>

The intermediate filament (IF) cytoskeleton is a general feature of differentiated cells. Its molecular components, IF proteins, constitute a large family including the evolutionarily conserved nuclear lamins and the more diverse collection of cytoplasmic intermediate filament (CIF) proteins. In vertebrates, genes encoding CIFs exhibit cell/tissue type-specific expression profiles and are thus useful as differentiation markers. The expression of invertebrate CIFs, however, is not well documented. Here, we report a whole-genome survey of IF genes and their developmental expression patterns in the leech Helobdella, a lophotrochozoan model for developmental biology research. We found that, as in vertebrates, each of the leech CIF genes is expressed in a specific set of cell/tissue types. This allows us to detect earliest points of differentiation for multiple cell types in leech development and to use CIFs as molecular markers for studying cell fate specification in leech embryos. In addition, to determine the feasibility of using CIFs as universal metazoan differentiation markers, we examined phylogenetic relationships of IF genes from various species. Our results suggest that CIFs, and thus their cell/tissue-specific expression patterns, have expanded several times independently during metazoan evolution. Moreover, comparing the expression patterns of CIF orthologs between two leech species suggests that rapid evolutionary changes in the cell or tissue specificity of CIFs have occurred among leeches. Hence, CIFs are not suitable for identifying cell or tissue homology except among very closely related species, but they are nevertheless useful species-specific differentiation markers.     

Epigenetic regulation of cell type-specific expression patterns in the human mammary epithelium

Differentiation is an epigenetic program that involves the gradual loss of pluripotency and acquisition of cell type-specific features. Understanding these processes requires genome-wide analysis of epigenetic and gene expression profiles, which have been challenging in primary tissue samples due to limited numbers of cells available. Here we describe the application of high-throughput sequencing technology for profiling histone and DNA methylation, as well as gene expression patterns of normal human mammary progenitor-enriched and luminal lineage-committed cells. We observed significant differences in histone H3 lysine 27 tri-methylation (H3K27me3) enrichment and DNA methylation of genes expressed in a cell type-specific manner, suggesting their regulation by epigenetic mechanisms and a dynamic interplay between the two processes that together define developmental potential. The technologies we developed and the epigenetically regulated genes we identified will accelerate the characterization of primary cell epigenomes and the dissection of human mammary epithelial lineage-commitment and luminal differentiation.     

Repositioning of muscle-specific genes relative to the periphery of SC-35 domains during skeletal myogenesis

Previous studies have shown that in a given cell type, certain active genes associate with SC-35 domains, nuclear regions rich in RNA metabolic factors and excluded from heterochromatin. This organization is not seen for all active genes; therefore, it is important to determine whether and when this locus-specific organization arises during development and differentiation of specific cell types. Here, we investigate whether gene organization relative to SC-35 domains is cell type specific by following several muscle and nonmuscle genes in human fibroblasts, committed but proliferative myoblasts, and terminally differentiated muscle. Although no change was seen for other loci, two muscle genes (Human beta-cardiac myosin heavy chain and myogenin) became localized to the periphery of an SC-35 domain in terminally differentiated muscle nuclei, but not in proliferative myoblasts or in fibroblasts. There was no apparent change in gene localization relative to either the chromosome territory or the heterochromatic compartment; thus, the gene repositioning seemed to occur specifically with respect to SC-35 domains. This gene relocation adjacent to a prominent SC-35 domain was recapitulated in mouse 3T3 cells induced into myogenesis by introduction of MyoD. Results demonstrate a cell type-specific reorganization of specific developmentally regulated loci relative to large domains of RNA metabolic factors, which may facilitate developmental regulation of genome expression.     

Cell type-specific filamin complex regulation by a novel class of HECT ubiquitin ligase is required for normal cell motility and patterning

Differential cell motility, which plays a key role in many developmental processes, is perhaps most evident in examples of pattern formation in which the different cell types arise intermingled before sorting out into discrete tissues. This is thought to require heterogeneities in responsiveness to differentiation-inducing signals that result in the activation of cell type-specific genes and 'salt and pepper' patterning. How differential gene expression results in cell sorting is poorly defined. Here we describe a novel gene (hfnA) that provides the first mechanistic link between cell signalling, differential gene expression and cell type-specific sorting in Dictyostelium. HfnA defines a novel group of evolutionarily conserved HECT ubiquitin ligases with an N-terminal filamin domain (HFNs). HfnA expression is induced by the stalk differentiation-inducing factor DIF-1 and is restricted to a subset of prestalk cells (pstO). hfnA(-) pstO cells differentiate but their sorting out is delayed. Genetic interactions suggest that this is due to misregulation of filamin complex activity. Overexpression of filamin complex members phenocopies the hfnA(-) pstO cell sorting defect, whereas disruption of filamin complex function in a wild-type background results in pstO cells sorting more strongly. Filamin disruption in an hfnA(-) background rescues pstO cell localisation. hfnA(-) cells exhibit altered slug phototaxis phenotypes consistent with filamin complex hyperactivity. We propose that HfnA regulates filamin complex activity and cell type-specific motility through the breakdown of filamin complexes. These findings provide a novel mechanism for filamin regulation and demonstrate that filamin is a crucial mechanistic link between responses to differentiation signals and cell movement in patterning based on 'salt and pepper' differentiation and sorting out.     

激光捕获显微切割技术在植物基因组研究中的应用

植物的生长和发育在很大程度上取决于组织和(或)器官特异表达的基因, 但要获取某一发育阶段的特异细胞类群来进行基因表达分析又是相当困难的。近年发展起来的激光捕获显微切割技术可以在显微镜下快速准确地获取单一的细胞类群, 甚至单个细胞, 成功地解决了组织中细胞的异质性问题。介绍了该技术的原理, 并对其在植物中的应用进展情况做了综述, 同时指出了该技术在植物中应用的可能发展方向。     

Isolation of developmentally regulated novel genes based on sequence identity and gene expression pattern.

Based on the surmise that a variety of genes might play important roles in embryonic development and tissue differentiation, and that some of them are likely to be expressed in undifferentiated ES cells, we attempted to identify new genes from the ES cell cDNA library. The modified method of expressed sequence tags (ESTs) and the examination of the expression patterns in adult tissues and in vitro differentiated ES cells were utilized in this study. We have isolated and identified several novel cDNA clones with interesting developmental expression pattern. Among the 83 clones randomly chosen, 23 clones (27.7%) have no homology to any sequences in public databases. The rest contain limited or complete sequence homology to the previously reported mammalian genes or ESTs, yet some clones have not been previously identified in the mouse. To examine the expression profile of clones during development and differentiation, sets of slot blots were hybridized with developmental stage specific or tissue specific probes. Out of 40 novel clones tested (21 totally unknown clones and 19 unidentified clones in mouse), most of them were up- or down-regulated as differentiation proceeded, and some clones showed differentiation-stage specific expression profiles. Surprisingly, a majority of genes were also expressed in adult tissues, and some clones even revealed tissue specific expression. These results demonstrate that not only was the strategy we employed in this study quite efficient for screening novel genes, but that the information gained by such studies would also be a useful guide for further analysis of these genes. It also suggests the feasibility of this approach to explore the genomewide network of gene expression during complicated biological processes, such as embryonic development and tissue differentiation.     

Gene Expression Signatures of Extracellular Matrix and Growth Factors during Embryonic Stem Cell Differentiation

Pluripotent stem cells are uniquely capable of differentiating into somatic cell derivatives of all three germ lineages, therefore holding tremendous promise for developmental biology studies and regenerative medicine therapies. Although temporal patterns of phenotypic gene expression have been relatively well characterized during the course of differentiation, coincident patterns of endogenous extracellular matrix (ECM) and growth factor expression that accompany pluripotent stem cell differentiation remain much less well-defined. Thus, the objective of this study was to examine the global dynamic profiles of ECM and growth factor genes associated with early stages of pluripotent mouse embryonic stem cell (ESC) differentiation. Gene expression analysis of ECM and growth factors by ESCs differentiating as embryoid bodies for up to 14 days was assessed using PCR arrays (172 unique genes total), and the results were examined using a variety of data mining methods. As expected, decreases in the expression of genes regulating pluripotent stem cell fate preceded subsequent increases in morphogen expression associated with differentiation. Pathway analysis generated solely from ECM and growth factor gene expression highlighted morphogenic cell processes within the embryoid bodies, such as cell growth, migration, and intercellular signaling, that are required for primitive tissue and organ developmental events. In addition, systems analysis of ECM and growth factor gene expression alone identified intracellular molecules and signaling pathways involved in the progression of pluripotent stem cell differentiation that were not contained within the array data set. Overall, these studies represent a novel framework to dissect the complex, dynamic nature of the extracellular biochemical milieu of stem cell microenvironments that regulate pluripotent cell fate decisions and morphogenesis.     

Detection of tissue-specific effects by methotrexate on differentiating mouse embryonic stem cells

BACKGROUND: Pluripotent embryonic stem (ES) cells offer a unique possibility to monitor the differentiation of several cell types in vitro. This study attempts to identify marker genes during in vitro cell differentiation of murine ES cells and allow a prediction of chemical effects on cell differentiation of specific target tissues. The study focused on the expression pattern of key genes involved in cardiomyocyte and osteoblast differentiation: Oct-4, Brachyury, Nkx2.5, alpha myosin heavy chain, Cbfa1, and Osteocalcin. METHODS: Methotrexate was selected due to its well-characterized teratogenic effects. Several in vivo studies have demonstrated the specific interactions of methotrexate with bone formation whereas the cardiovascular system is not specifically affected after exposure to low concentration. The capability of murine ES cells to differentiate in vitro into cardiomyocytes as well as into osteoblasts have been used to demonstrate the target cell specificity in vitro, at non-cytotoxic concentration. RESULTS: Exposure of differentiating ES cells did not result in any gene profile modification of the selected cardiomyocyte specific genes, whereas the expression of osteoblast specific key genes, Cbfa1 and Osteocalcin, decreased. At the latter stages of skeletal differentiation we observed a 30% decrease in gene expression for Cbfa1 and a 60% decrease for Osteocalcin, with reference to the control. Early marker genes for undifferentiated cells and mesodermal cells were not modified after methotrexate treatment. CONCLUSIONS: These results show the possibility to integrate specific in vitro tests for teratogenicity in a test strategy for developmental toxicity.     

Ectopic synthesis of epidermal cytokeratins in pancreatic islet cells of transgenic mice interferes with cytoskeletal order and insulin production

The members of the multigene family of intermediate filament (IF) proteins are expressed in various combinations and amounts that are specific for a given pathway or state of differentiation. Previous experiments in which the cell type-specific IF cytoskeleton was altered by introducing foreign IF proteins into cultured cells or certain tissues of transgenic animals have shown a remarkable tolerance, without detectable interference with cell functions. To examine the importance of the cell type-specific cytokeratin (CK) IF pattern, we have studied the ectopic expression of CK genes in different epithelia of transgenic mice. Here we report changes observed in the beta cells of pancreatic islets expressing the genes for human epidermal CKs 1 and/or 10 brought under control of the rat insulin promoter. Both genes were efficiently expressed, resulting in the appearance of numerous and massive bundles of aggregated IFs, resembling those of epidermal keratinocytes. While the synthesis of epidermal CK 10 was readily accommodated and compatible with cell function, mice expressing CK 1 in their beta cells, alone or in combination with CK 10, developed a special form of diabetes characterized by a drastic reduction of insulin-secretory vesicles and of insulin-and CK 1-producing cells. In many CK 1-producing cells, accumulations of fibrous or granular material containing CK 1 were also seen in the nucleus. This demonstration of functional importance of the specific CK-complement in an epithelial cell indicates a contribution of cell type-specific factors to cytoplasmic IF compartmentalization and that the specific CK complement can be crucial for functions and longevity of a given kind of epithelium.     

Intrinsic properties and external factors determine the differentiation bias of human embryonic stem cell lines

A major goal of human embryonic stem cell (hESC) research is to regulate differentiation through external means to generate specific cell types with high purity for regenerative medicine applications. Although all hESC lines express pluripotency‐associated genes, their differentiation ability to various lineages differs considerably. We have compared spontaneous differentiation propensity of the two hESC lines, RelicellhES1 and BG01. Spontaneous differentiation of hESC lines grown in different media conditions was followed by differentiation using two methods. Kinetic data generated by real‐time gene expression studies for differentiated cell types were analyzed, and confirmed at protein levels. Both cell lines showed upregulation of genes associated with the 3 germ layers, although stark contrast was evident in the magnitude of upregulation of lineage specific genes. A distinct difference was also found in the rate at which the pluripoteny factors, Oct‐4 and Nanog, were downregulated during differentiation. Once differentiation was initiated, both Oct‐4 and Nanog gene expression was drastically reduced in RelicellhES1, whereas a gradual decrease was observed in BG01. A clear trend is seen in RelicellhES1 to differentiate into neuroectodermal and mesenchymal lineages, whereas BG01 cells are more prone to mesoderm and endoderm development. In addition, suspension versus plated methods of cell culture significantly influenced the outcome of differentiation of certain types of cells. Results obtained by spontaneous differentiation of hESCs were also amplified by induced differentiation. Thus, differential rates of downregulation of pluripotency markers along with culture conditions seem to play an important role in determining the developmental bias of human ES cell lines.