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Previous studies, particularly in amphibians, have demonstrated that the initial step of vertebrate neural differentiation from uncommitted embryonic ectodermal cells occurs via a cell-intrinsic mechanism and requires only that inhibitory [...], The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.12.016 Byline: Atsushi Takebe (a)(c), Takumi Era (a), Mitsuhiro Okada (a)(b), Lars Martin Jakt (a)(b), Yoshikazu Kuroda (c), Shin-Ichi Nishikawa (a) Keywords: ES cell; In vitro differentiation; DNA microarray; PDGFR[alpha]; ARID3b Abstract: An inherent difficulty in using DNA microarray technology on the early mouse embryo is its relatively small size. In this study, we investigated whether use of ES cell differentiation culture, which has no theoretical limit in the number of cells that can be generated, can improve this situation. Seven distinct ES-cell-derived populations were analyzed by DNA microarray and examined for genes whose distribution patterns are similar to those of PDGFR[alpha], a gene implicated in differentiation of mesoderm/mesenchymal lineages. Using software developed in our laboratory, we formed a group of 30 genes which showed the highest similarity to PDGFR[alpha], 18 of these genes were shown to be involved in development of either mesodermal, mesenchymal or neural crest cells. This list also contains several genes whose role in embryogenesis has not yet been fully identified. One such molecule is mARID3b. The mARID3b expression is found in the paraxial mesoderm and cranial mesenchyme. mARID3b-null mouse showed early embryonic lethality, and most phenotypes of this mutant appear to develop from a failure to generate a sufficient number of cranial mesenchymal cells. These results demonstrate the potential use of ES cell differentiation culture in identifying novel genes playing an indispensable role in embryogenesis. Author Affiliation: (a) Laboratory for Stem Cell Biology, RIKEN Center for Development Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan (b) The Foundation for Biomedical Research and Innovation, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan (c) Division of Gastroenterological Surgery, Graduate School of Medicine, Kobe University, 7-5-2 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan Article History: Received 27 June 2005; Revised 18 November 2005; Accepted 6 December 2005

Identification of novel genes that are similar to platelet-derived growth factor receptor, using DNA microarray studies on mouse embryonic cells, to find their role in embryogenesis is presented.

Diet has been shown to influence epigenetic key players, such as DNA methylation, which can regulate the gene expression potential in both parents and offspring. Diets enriched in omega-6 and deficient in omega-3 PUFAs (low dietary omega-3/omega-6 PUFA ratio), have been associated with the promotion of pathogenesis of diseases in humans and other mammals. In this study, we investigated the impact of increased dietary intake of arachidonic acid (ARA), a physiologically important omega-6 PUFA, on 2 generations of zebrafish. Parental fish were fed either a low or a high ARA diet, while the progeny of both groups were fed the low ARA diet. We screened for DNA methylation on single base-pair resolution using reduced representation bisulfite sequencing (RRBS). The DNA methylation profiling revealed significant differences between the dietary groups in both parents and offspring. The majority of differentially methylated loci associated with high dietary ARA were found in introns and intergenic regions for both generations. Common loci between the identified differentially methylated loci in F0 and F1 livers were reported. We described overlapping gene annotations of identified methylation changes with differential expression, but based on a small number of overlaps. The present study describes the diet-associated methylation profiles across genomic regions, and it demonstrates that parental high dietary ARA modulates DNA methylation patterns in zebrafish liver. [ABSTRACT FROM AUTHOR]

A heteroplasmic tandem repeat (HTR) array occupies 100 to 300 bp of the mitochondrial DNA control region in the Atlantic cod, and recently we noted that the repeat appeared integrated in a polyadenylated mitochondrial long noncoding RNA. Here we provide a more detailed analysis of the mitochondrial HTR in the mitochondrial genome of 134 Atlantic cod specimens. We report all specimens to harbor mitochondrial HTRs in the control region, and identified 26 distinct variants among the 402 repeat motifs assessed. Whereas most specimens contained HTR profiles of 2-5 copies consisting of the same 40-bp motif, 22 specimens showed compound HTR arrays of at least two types of motifs present in the same mitochondrial DNA molecule. We found HTR profiles to be highly conserved between different tissue types of a single individual, and strictly maternally inherited in a mating experiment between parental Atlantic cod expressing different HTR profiles and array motifs. We conclude that mitochondrial heteroplasmy in the control region is very common in Atlantic cod, and results in length heterogenity of the long noncoding RNA lncCR-H. [ABSTRACT FROM AUTHOR]

The field of hematopoietic and vascular developmental research owes its origin to the chick embryo. Many key concepts, such as the hematopoietic stem cell, hemangioblast and hemogenic endothelium, were first proposed in this model organism. Genetically tractable models have gradually replaced the chick in the past two decades. However, advances in comparative genomics, transcriptomics and promoteromics promise a re-emergence of the chick embryo as a powerful model for hematopoietic/vascular research. This review summarizes the current status of our understanding of early blood/vascular development in the chick, focusing primarily on the processes of primitive hematopoiesis and early vascular network formation in the extraembryonic and lateral plate mesoderm territories. Emphasis is given to ontological and molecular association between the blood and endothelial cells and to the evolutionary relationship between the hemangioblasts, common precursors for the blood and endothelial lineages, and the coelomic epithelial lining cells. Links between early blood/vascular development and later definitive hematopoiesis are also discussed. Finally, potential applications of the chick model for comparative and omics-level studies of the blood/vascular system are highlighted. [ABSTRACT FROM AUTHOR]

DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells. [ABSTRACT FROM AUTHOR]

Oligonucleotide-based fluorescent in situ hybridization (FISH) coupled with high-resolution high-sensitivity microscopy allows the visualization of single RNA molecules within fixed cells and tissues as distinct foci. We show here that combinatorial labeling of RNA molecules with several fluorescent dyes extends the number of genes that can be targeted simultaneously beyond the number of fluorophores used. This approach also inherently validates the identification of transcripts reducing false positive counts. We have used combinatorial FISH and image analysis to measure the transcript densities of six genes using three fluorophores. This has allowed us to visualize the endothelial maturation of lateral mesoderm in an in vitro ES differentiation assay from a single snapshot of molecular identities. Our observations show that, under these specific conditions, endothelial maturation follows a homogeneous course with a gradual increase in expression of Cdh5 and a concomitant loss of early transcription factors, arguing that maturation is governed in a generally deterministic manner. This methodology is limited by the number of fluorophores that can be used and by the available microscopic resolution, but currently available equipment should allow the visualization of transcripts from 10 or more genes simultaneously. [ABSTRACT FROM AUTHOR]

Background: Drugs inhibiting vascular endothelial growth factor (VEGF) signaling are globally administered to suppress deregulated angiogenesis in a variety of eye diseases. However, anti-VEGF therapy potentially affects the normal functions of retinal neurons and glias which constitutively express VEGF receptor 2. Thus, it is desirable to identify novel drug targets which are exclusively expressed in endothelial cells (ECs). Here we attempted to identify an EC-specific Rho guanine nucleotide exchange factor (GEF) and evaluate its role in retinal angiogenesis. Methodology/Principal Findings: By exploiting fluorescence-activated cell sorting and microarray analyses in conjunction with in silico bioinformatics analyses, we comprehensively identified endothelial genes in angiogenic retinal vessels of postnatal mice. Of 9 RhoGEFs which were highly expressed in retinal ECs, we show that Arhgef15 acted as an EC-specific GEF to mediate VEGF-induced Cdc42 activation and potentiated RhoJ inactivation, thereby promoting actin polymerization and cell motility. Disruption of the Arhgef15 gene led to delayed extension of vascular networks and subsequent reduction of total vessel areas in postnatal mouse retinas. Conclusions/Significance: Our study provides information useful to the development of new means of selectively manipulating angiogenesis without affecting homeostasis in un-targeted tissues; not only in eyes but also in various disease settings such as cancer. [ABSTRACT FROM AUTHOR]

The cell biology of the early processes of mammalian embryogenesis, such as germ-layer formation, has been technically challenging to study owing to the size and accessibility of mammalian embryos. Embryonic stem cells, which can generate the three germ layers in vitro, are useful for studying embryogenesis at the cellular level. So, how can the study of embryonic stem cells and their differentiation provide a deeper understanding of the cell biology of early development? [ABSTRACT FROM AUTHOR]

ALCAM (activated leukocyte cell adhesion molecule, CD166) belongs to the immunoglobulin superfamily and is involved in axon guidance, hematopoiesis, immune response and tumor metastasis. During embryogenesis, mRNA encoding ALCAM was expressed in the cardiac crescent and the neural groove at embryonic day (E) 7.75 and predominately in the tubular heart at E8.5. A newly generated monoclonal antibody against the ALCAM molecule (ALC-48) exclusively stained cardiomyocytes at E8.25–10.5. However, ALCAM expression was lost by cardiomyocytes by E12.5 and its expression shifts to a variety of organs during later stages. ALCAM was found to be a prominent surface marker for cardiomyocytes in early embryonic hearts. The transient expression of ALCAM during early developmental stages marks specific developmental stages in cardiomyocyte differentiation. Copyright © 2007 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Preparation of specific lineages at high purities from embryonic stem (ES) cells requires both selective culture conditions and markers to guide and monitor the differentiation. In this study, we distinguished definitive and visceral endoderm by using a mouse ES cell line that bears the gfp and human IL2Rα (also known as CD25) marker genes in the goosecoid (Gsc) and Sox17 loci, respectively. This cell line allowed us to monitor the generation of Gsc+Sox17+ definitive endoderm and Gsc−Sox17+ visceral endoderm and to define culture conditions that differentially induce definitive and visceral endoderm. By comparing the gene expression profiles of definitive and visceral endoderm, we identified seven surface molecules that are expressed differentially in the two populations. One of the seven markers, Cxcr4, to which a monoclonal antibody is available allowed us to monitor and purify the Gsc+ population from genetically unmanipulated ES cells under the condition that selects definitive endoderm. [ABSTRACT FROM AUTHOR]

Treatment with the demethylating drugs 5-azacytidine (AZA) and decitabine (DAC) is now recognised as an effective therapy for patients with Myelodysplastic Syndromes (MDS), a range of disorders arising in clones of hematopoietic progenitor cells. A variety of cell models have been used to study the effect of these drugs on the methylation of promoter regions of tumour suppressor genes, with recent efforts focusing on the ability of these drugs to inhibit DNA methylation at low doses. However, it is still not clear how nano-molar drug treatment exerts its effects on the methylome. In this study, we have characterised changes in DNA methylation caused by prolonged low-dose treatment in a leukemic cell model (SKM-1), and present a genome-wide analysis of the effects of AZA and DAC. At nano-molar dosages, a one-month continuous treatment halved the total number of hypermethylated probes in leukemic cells and our analysis identified 803 candidate regions with significant demethylation after treatment. Demethylated regions were enriched in promoter sequences whereas gene-body CGIs were more resistant to the demethylation process. CGI methylation in promoters was strongly correlated with gene expression but this correlation was lost after treatment. Our results indicate that CGI demethylation occurs preferentially at promoters, but that it is not generally sufficient to modify expression patterns, and emphasises the roles of other means of maintaining cell state. [ABSTRACT FROM AUTHOR]

From a list of protein kinases (PKs) that are newly induced upon differentiation of mouse embryonic stem cells to mesendoderm, we identified a previously uncharacterized kinase, Vlk (vertebrate lonesome kinase), that is well conserved in vertebrates but has no homologs outside of the vertebrate lineage. Its kinase domain cannot be classified into any of the previously defined kinase groups or families. Although Vlk is first expressed in E-cadherin-positive anterior visceral endoderm and mesendoderm, its expression is later confined to E-cadherin-negative mesenchyme. Vlk is enriched in the Golgi apparatus and blocks VSVG transport from the Golgi to the plasma membrane. Targeted disruption of Vlk leads to a defect in lung development and to delayed ossification of endochondral bone. Vlk-/- mice display neonatal lethality due to respiratory failure, with a suckling defect arising from a cleft palate. Our results demonstrate that Vlk is a novel vertebrate-specific PK that is involved in the regulation of the rate of protein export from the Golgi, thereby playing an important role in the formation of functional stroma by mesenchymal cells. [ABSTRACT FROM AUTHOR]

Genome studies in fish provide evidence for the adaptability of the vertebrate immune system, revealing alternative immune strategies. The reported absence of the major compatibility complex (MHC) class II pathway components in certain species of pipefish (genus Syngnathus ) and cod-like fishes (order Gadiformes) is of particular interest. The MHC II pathway is responsible for immunization and defence against extracellular threats through the presentation of exogenous peptides to T helper cells. Here, we demonstrate the absence of all genes encoding MHC II components (CD4, CD74 A/B, and both classical and non-classical MHC II α / β ) in the genome of an anglerfish, Lophius piscatorius , indicating loss of the MHC II pathway. By contrast, it has previously been reported that another anglerfish, Antennarius striatus , retains all MHC II genes, placing the loss of MHC II in the Lophius clade to their most recent common ancestor . In the three taxa where MHC II loss has occurred, the gene loss has been restricted to four or five core MHC II components, suggesting that, in teleosts, only these genes have functions that are restricted to the MHC II pathway.

Micronutrient status of parents can affect long term health of their progeny. Around 2 billion humans are affected by chronic micronutrient deficiency. In this study we use zebrafish as a model system to examine morphological, molecular and epigenetic changes in mature offspring of parents that experienced a one-carbon (1-C) micronutrient deficiency. Zebrafish were fed a diet sufficient, or marginally deficient in 1-C nutrients (folate, vitamin B12, vitamin B6, methionine, choline), and then mated. Offspring livers underwent histological examination, RNA sequencing and genome-wide DNA methylation analysis. Parental 1-C micronutrient deficiency resulted in increased lipid inclusion and we identified 686 differentially expressed genes in offspring liver, the majority of which were downregulated. Downregulated genes were enriched for functional categories related to sterol, steroid and lipid biosynthesis, as well as mitochondrial protein synthesis. Differential DNA methylation was found at 2869 CpG sites, enriched in promoter regions and permutation analyses confirmed the association with parental feed. Our data indicate that parental 1-C nutrient status can persist as locus specific DNA methylation marks in descendants and suggest an effect on lipid utilization and mitochondrial protein translation in F 1 livers. This points toward parental micronutrients status as an important factor for offspring health and welfare.

The field of hematopoietic and vascular developmental research owes its origin to the chick embryo. Many key concepts, such as the hematopoietic stem cell, hemangioblast and hemogenic endothelium, were first proposed in this model organism. Genetically tractable models have gradually replaced the chick in the past two decades. However, advances in comparative genomics, transcriptomics and promoteromics promise a re-emergence of the chick embryo as a powerful model for hematopoietic/vascular research. This review summarizes the current status of our understanding of early blood/vascular development in the chick, focusing primarily on the processes of primitive hematopoiesis and early vascular network formation in the extraembryonic and lateral plate mesoderm territories. Emphasis is given to ontological and molecular association between the blood and endothelial cells and to the evolutionary relationship between the hemangioblasts, common precursors for the blood and endothelial lineages, and the coelomic epithelial lining cells. Links between early blood/vascular development and later definitive hematopoiesis are also discussed. Finally, potential applications of the chick model for comparative and omics-level studies of the blood/vascular system are highlighted.

Myelodysplastic syndrome (MDS) is a disorder of hematopoietic stem cells (HSCs) that is often treated with DNA methyltransferase 1 (DNMT1) inhibitors (5-azacytidine [AZA], 5-aza-2'-deoxycytidine), suggesting a role for DNA methylation in disease progression. How DNMT inhibition retards disease progression and how DNA methylation contributes to MDS remain unclear. We analyzed global DNA methylation in purified CD34+ hematopoietic progenitors from MDS patients undergoing multiple rounds of AZA treatment. Differential methylation between MDS phenotypes was observed primarily at developmental regulators not expressed within the hematopoietic compartment and was distinct from that observed between healthy hematopoietic cell types. After AZA treatment, we observed only limited DNA demethylation at sites that varied between patients. This suggests that a subset of the stem cell population is resistant to AZA and provides a basis for disease relapse. Using gene expression data from patient samples and an in vitro AZA treatment study, we identified differentially methylated genes that can be activated following treatment and that remain silent in the CD34+ stem cell compartment of high-risk MDS patients. Haploinsufficiency in mice of one of these genes (NR4A2) has been shown to lead to excessive HSC proliferation, and our data suggest that suppression of NR4A2 by DNA methylation may be involved in MDS progression., (©AlphaMed Press.)

Expression of the Wnt modulator secreted frizzled related protein 4 (Sfrp4) is upregulated after heart ischemic injury. We show that intramuscular administration of recombinant Sfrp4 to rat heart ischemic injury and recanalization models prevents further deterioration of cardiac function after the ischemic injury. The effect of Sfrp4 persisted for at least 20 weeks when Sfrp4 was administered in a slow release system (Sfrp4-polyhedra) to both acute and subacute ischemic models. The histology of the dissected heart showed that the cardiac wall was thicker and the area of acellular scarring was smaller in Sfrp4-treated hearts than in controls. Increased amounts of both the inactive serine 9-phosphorylated form of glycogen synthase kinase (GSK)-3β and the active form of β-catenin were observed by immunohistology 3 days after lateral anterior descendant ligation in control, but not in Sfrp4-treated hearts. All together, we show that administration of Sfrp4 interferes with canonical Wnt signaling that could mediate the formation of acellular scar and consequently contributes to the prevention of aggravation of cardiac function.

ARID3B, a member of the AT-rich interaction domain (ARID) family of proteins, plays an essential role in the survival of neural crest during embryogenesis. Here, we report evidence that ARID3B is involved in the development of malignant neuroblastoma, a childhood tumor derived from neural crest. (a) ARID3B is expressed by all five cell lines derived from neuroblastoma tested by us. (b) Analysis of published DNA microarray data of fresh neuroblastoma tumors showed that ARID3B is expressed in 80% of stage IV tumors, whereas only in 9% of stage I-III+IVs tumors. (c) In vitro growth of several neuroblastoma cell lines is suppressed significantly by antisense as well as siRNA treatment. (d) An increase of the ARID3B expression level by transfection in the SY5Y neuroblastoma cell line enhances the malignancy in tumor growth assays in nu/nu mice. (e) ARID3B by itself can immortalize mouse embryonic fibroblasts (MEFs) in vitro and confers malignancy to MEF when transfected together with MYCN, the best characterized oncogene for neuroblastoma. Thus, ARID3B seems to play a key role in the malignant transformation of neuroblastoma and may serve not only as a marker of malignancy but also as a potential target for cancer therapy of stage IV neuroblastoma for which there is currently no effective treatment available.

Endothelial cells (ECs) are thought to be derived mainly from the vascular endothelial growth factor receptor 2 (VEGFR-2)+ lateral mesoderm during early embryogenesis. In this study, we specified several pathways for EC differentiation using a murine embryonic stem (ES) cell differentiation culture system that is a model for cellular processes during early embryogenesis. Based on the results of in vitro fate analysis, we show that, in the main pathway, committed ECs are differentiated through the VEGFR-2+ platelet-derived growth factor receptor alpha (PDGFR-alpha)- single-positive (VSP) population that is derived from the VEGFR-2+ PDGFR-alpha+ double-positive (DP) population. This major differentiation course was also confirmed using DNA microarray analysis. In addition to this main pathway, however, ECs also can be generated from the VEGFR-2- PDGFR-alpha+ single-positive (PSP) population, which represents the paraxial mesodermal lineage and is also derived from the DP population. Our results strongly suggest that, even after differentiation from the common progenitor DP population into the VSP and PSP populations, these two populations continue spontaneous switching of their surface phenotype, which results in switching of their eventual fates. The rate of this interlineage conversion between VSP and PSP is unexpectedly high. Because of this potential to undergo fate switch, we conclude that ECs can be generated via multiple pathways in in vitro ES cell differentiation.