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PubMed2004

Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns.

Genetic approaches in Drosophila led to the identification of Forkhead, the prototype of forkhead domain transcription factors that are now known to comprise an evolutionarily conserved family of proteins with essential roles in development and differentiation. Sequence analysis of the recently published genomic scaffold sequence from Drosophila melanogaster has allowed us to determine the presumably full complement of forkhead domain encoding genes in this species. We show herein that the Drosophila genome contains 17 forkhead domain encoding genes; 13 of these genes have orthologs in chordate species, and their products can be assigned to 10 of the 17 forkhead domain subclasses known from chordates. One Drosophila forkhead domain gene only has a Caenorhabditis elegans ortholog and may represent a subclass that is absent in chordates, while the remaining three cannot be classified. We present the mRNA expression patterns of seven previously uncharacterized members of this gene family and show that they are expressed in tissues from all three germ layers, including central and peripheral nervous system, epidermis, salivary gland primordia, endoderm, somatic mesoderm, and hemocyte progenitors. Furthermore, the expression patterns of two of these genes, fd19B and fd102C, suggest a role for them as gap genes during early embryonic head segmentation.

Amino Acid Sequence↗
PubMed2001

Notochord patterning of the endoderm.

Endodermally derived organs of the gastrointestinal and respiratory system form at distinct anterioposterior and dorsoventral locations along the vertebrate body axis. This stereotyped program of organ formation depends on the correct patterning of the endodermal epithelium so that organ differentiation and morphogenesis occur at appropriate positions along the gut tube. Whereas some initial patterning of the endoderm is known to occur early, during germ-layer formation and gastrulation, later signaling events, originating from a number of adjacent tissue layers, are essential for the development of endodermal organs. Previous studies have shown that signals arising from the notochord are important for patterning of the ectodermally derived floor plate of the neural tube and the mesodermally derived somites. This review will discuss recent evidence indicating that signals arising from the notochord also play a role in regulating endoderm development.

Body Patterning↗
PubMed2025

Enhancer remodeling by OTX2 directs specification and patterning of mammalian definitive endoderm.

The molecular mechanisms that drive essential patterning events in the mammalian embryo remain poorly understood. Analysis of transcription factor expression kinetics at peri-gastrulation stages of development suggest Otx2 as a candidate regulator of the definitive endoderm, the precursor of all gut-derived organs. Accordingly, timed OTX2 depletion in gastruloids or during directed differentiation results in abnormal definitive endoderm specification in mouse and human, characterized by altered expression of components and transcriptional targets of the canonical WNT signaling pathway, perturbed adhesion and migration programs, and de-repression of regulators of other lineages. These defects cumulate in impaired foregut formation. Mechanistically, OTX2 is required to activate a subset of endoderm-specific enhancers and to suppress select enhancers of other lineages, allowing timely exit from the primitive streak and correct specification of anterior endoderm. Our results establish OTX2 as an early gut regulator and suggest molecular principles underlying spatiotemporal cell identity conserved across germ layers and species.

Otx Transcription Factors↗
PubMed1981

Reactivity of monoclonal antibodies against intermediate filament proteins during embryonic development.

Monoclonal antibodies (mAbs) against a preparation of intermediate filaments from trophoblastoma cells were studied for their reactivity pattern during embryonic development and on adult tissue cells. Up to day 12 of embryonic development, epithelial cells of the three germ layers reacted with these mAbs. Later during development and in adult tissues, positive reactions could be observed only with epithelial cells derived from mesoderm and endoderm. Because of their tissue distribution, the proteins reacting with these mAbs might belong to the keratin family of intermediate filaments or they might represent a new group of intermediate filaments.

Animals↗
PubMed2004

[Progress on embryonic stem cells for the treatment of myocardial infarction].

Embryonic stem cells are pluripotent cells derived from blastocyst-stage embryos. They are characterized by their capacity for self-renewal and by the ability to differentiate into a variety of cell types. In vitro, ES cells can spontaneously aggregate into the formation of embryoid bodies (EBs) comprised of a range of differentiated cells. When injected into severe combined immunodeficient (SCID) mice, ES cells will induce the generation of teratomas, which include differentiated cells from three embryonic germ layers. We can induce the differentiation of ES cells into cardiocytes by treatment with several growth factors or co-culture with certain other cells. The pure differentiated cardiocytes from ES cells are then selected and implanted into the infarcted hearts. The hearts' function will be improved. ES cells implantation is a novel and potential method to treat infarcted hearts.

Animals↗
PubMed2002

Monkey embryonic stem cell lines expressing green fluorescent protein.

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

Animals↗
PubMed1983

Histogenetic and neoplastic potential of different regions of the mouse embryonic egg cylinder.

The histogenetic and neoplastic potentials of defined regions of the 8th day mouse embryonic egg cylinder were examined following ectopic transfer to beneath the testis capsule. No differences in histogenetic potential were detected between anterior and posterior slices of the embryo, either when composed of all three germ layers or of embryonic ectoderm alone. Small anterior and distal fragments of embryonic ectoderm also produced similar histogenetic profiles, although posterior fragments failed to grow in this ectopic site. The histogenetic potential of anterior and distal fragments exceeded the developmental fate ascribed to these two regions in the embryo (Beddington, 1981). There was some evidence for regionalization with respect to neoplastic potential, anterior slices of the embryo giving rise to a higher incidence of embryonal carcinoma cells than posterior slices.

Animals↗
PubMed2003

Regulatory signals and tissue interactions in the early hematopoietic cell differentiation in Xenopus laevis embryo.

Bone morphogenetic protein-4 (BMP-4) has been shown as an essential factor in differentiation of the primitive blood cells in Xenopus laevis embryo. Organizer factors, in contrast, function as a negative regulator for the blood cell differentiation. Differentiation of both blood cells and muscle tissue are negatively regulated by the organizer activity. However, blood cells but not muscle tissue can differentiate in the organizer-depleted embryos produced by removal of vegetal cortex cytoplasm at the one-cell stage. Thus the blood cells are totally independent cells from the organizer activity. The down-stream target molecules of the BMP-4 signaling, such as vent-1/2 and msx-1/2 are the positive regulators for muscle tissue differentiation, whereas these factors do not promote blood cell formation. It has not yet been elucidated how the BMP-4 signaling promotes the differentiation of blood cells, but it is likely that transcription factors such as GATA-2, SCL, LMO-2, biklf and GATA-1 are at least involved in the initial blood program. Experiments using combined germ layer explants suggest that interaction between ectoderm and mesoderm at the gastrula stage is important for the blood cell formation in mesoderm. BMP-4 produced in the ectodermal cells is essential for this interaction. For understanding the whole program in the embryonic blood cell differentiation, it is important to elucidate the molecular mechanisms underlying the tissue-tissue interaction, in addition to the analysis of the regulatory cascade that takes place in the mesodermal cells.

Animals↗
PubMed1994

Retroviral-mediated gene transfer into early passage cultures of ovine neuroectodermal, epithelial and mesenchymal tissues.

The relative efficiency of retroviral-mediated gene transfer into early passage cultures of different tissues of fetal lamb was investigated. Cells which originate from different embryonic germ layers showed marked differences in infectibility in the descending order: brain (neuroectoderm) > kidney (nephrogenic mesoderm) > muscle (somite mesoderm), lung (endoderm) > skin (ectoderm). In an attempt to generate immortalized brain cell lines, primary cultures were transduced with various oncogenes. A wide variety of morphologically distinct, dividing cells, including neurone-like and glial-like cells, were obtained using some oncogenes but also using the vector without an inserted oncogene.

Animals↗
PubMed2001

Expression of the human NOV gene in first trimester fetal tissues.

NOV, located on human chromosome 8q24.1, was originally cloned following discovery of its avian homolog as a consequence of over-expression in virally induced nephroblastoma. The gene product is a secreted, modular, protein and a member of the CCN gene family. Evidence to date indicates that the expression of the wild type protein is associated with cellular quiescence in normal embryonic fibroblasts yet produces growth stimulatory effects on established murine NIH 3T3 cells. Here we report the expression of NOV in the first trimester of human embryogenesis, between 5 and 10 weeks. In situ hybridisation and immunohistochemistry reveal widespread expression in derivatives of all three germ layers. The most abundant sites of expression are in the motor neurons and floor plate of the spinal cord, adrenal cortex, fusing skeletal, and smooth muscle, the urogenital system and the developing heart. Additionally, expression is seen in the cranial ganglia, differentiating chondrocytes, gonads, and lung. The sites of expression suggest strongly that autocrine or paracrine expression of NOV is associated with the process of cell differentiation.

Adrenal Cortex↗
PubMed1997

Self renewal of embryonic stem cells in the absence of feeder cells and exogenous leukaemia inhibitory factor.

To evaluate the role of leukaemia inhibitory factor (LIF) for maintaining pluripotent embryonic stem (ES) cells in culture, we established several exogenous LIF-independent ES cell lines by continuous passaging in culture. The newly established ES cells, Kli and CBli, sustained their growth and remained undifferentiated in LIF-deficient medium. Analysis of chimaeric animals, produced with the beta-galactosidase transgenic Kli ES cells, revealed that LIF-independent ES cells can contribute to all embryonic germ layers. There was no detectable LIF protein in ES cell conditioned medium, and no upregulation of LIF mRNA was found. The addition of neutralising anti-LIF antibodies was not sufficient to abrogate the self renewal of the Kli ES cells. These studies suggest that the signalling pathway involving diffusible LIF can be bypassed for maintaining the pluripotency in culture, and indicate a considerable heterogeneity in growth factor dependence and differentiation of different ES cells.

Animals↗
PubMed2000

Isolation of pluripotent embryonic stem cells from reprogrammed adult mouse somatic cell nuclei.

Pluripotent human stem cells isolated from early embryos represent a potentially unlimited source of many different cell types for cell-based gene and tissue therapies [1-3]. Nevertheless, if the full potential of cell lines derived from donor embryos is to be realised, the problem of donor-recipient tissue matching needs to be overcome. One approach, which avoids the problem of transplant rejection, would be to establish stem cell lines from the patient's own cells through therapeutic cloning [3,4]. Recent studies have shown that it is possible to transfer the nucleus from an adult somatic cell to an unfertilised oocyte that is devoid of maternal chromosomes, and achieve embryonic development under the control of the transferred nucleus [5-7]. Stem cells isolated from such a cloned embryo would be genetically identical to the patient and pose no risk of immune rejection. Here, we report the isolation of pluripotent murine stem cells from reprogrammed adult somatic cell nuclei. Embryos were generated by direct injection of mechanically isolated cumulus cell nuclei into mature oocytes. Embryonic stem (ES) cells isolated from cumulus-cell-derived blastocysts displayed the characteristic morphology and marker expression of conventional ES cells and underwent extensive differentiation into all three embryonic germ layers (endoderm, mesoderm and ectoderm) in tumours and in chimaeric foetuses and pups. The ES cells were also shown to differentiate readily into neurons and muscle in culture. This study shows that pluripotent stem cells can be derived from nuclei of terminally differentiated adult somatic cells and offers a model system for the development of therapies that rely on autologous, human pluripotent stem cells.

Animals↗
PubMed2004

Nr0b1 and its network partners are expressed early in murine embryos prior to steroidogenic axis organogenesis.

Ahch is an orphan nuclear receptor encoded by Nr0b1 on the murine X chromosome and is the ortholog of human DAX1. Nr0b1/NR0B1 expression at appropriate dosages is required for normal steroidogenic axis development: mutation of the human ortholog, NR0B1, results in adrenal hypoplasia congenita and hypogonadotropic hypogonadism; and duplication or transgenic overexpression in humans or mice, respectively, results in XY phenotypic females, a phenotype known as dosage sensitive sex-reversal. Complete loss of Nr0b1 by targeted deletion has been hypothesized to be lethal in embryonic stem (ES) cells and preliminary evidence suggested that ES cells might express Nr0b1. These investigations examined Nr0b1 expression and its network partners in both cultured ES cells and preimplantation embryos. We cultured ES cells in the absence or presence of differentiation agents and analyzed expression of Nr0b1 and associated network partners by northern blot hybridization and reverse transcriptase-polymerase chain reaction. Nrob1 was highly expressed by totipotent ES cells with reduced expression following induction toward individual germ layer fates. Nr5a1/Sf1, Wt1 and other genes that encode proteins known to interact with Nr0b1 were also expressed. Immunohistochemical analysis of preimplantation embryos for Ahch and key partners confirmed in vivo expression of network components. These findings are consistent with the existence of a potentially functional network of transcription factors, including Ahch, very early in embryonic development. These results validate ES cells as a developmentally dynamic model for mechanistic investigations into this regulatory network early in embryogenesis preceding organogenesis.

Animals↗
PubMed1982

Immunocytochemical localisation of alpha-fetoprotein (AFP) and serum albumin (ALB) in ecto-, meso- and endodermal tissue derivatives of the developing rat.

Recent work has provided evidence of the intracellular presence of AFP and serum albumin (ALB) in the central nervous system of rats, mice and primates. We report here the immunocytochemical localization of these proteins in tissue derivatives of the three germ layers (with the exclusion of nervous system components) during rat fetal and neonatal development. Positive labelling for AFP and ALB was transitorily observed in all tissue derivatives examined. For example, labelling for AFP in the epidermis started around the 16th day of fetal life and localized in some cell aggregations of the peridermis. Between the 17th and the 19th day AFP positive cells were observed in the basal layer of epidermis, while the stratum germinativum was always negative. The maximum intensity of labelling was at birth. At this time, AFP positive cells were visible in the strata spinosum, granulosum and corneum. On day 6 to 8 after birth, the labelling become negative. The localisation of ALB showed a similar pattern with the only difference of a short (2 to 5 days) displacement in time. It can be concluded that the intracellular uptake of AFP in the structures studied is transitory and follows the stage of cell and tissue differentiation. Relative to AFP, labelling for ALB seems to correspond to a more advanced stage of morphologic development.

Animals↗
PubMed2006

Transduction of human embryonic stem cells by ecotropic retroviral vectors.

The steadily increasing availability of human embryonic stem (hES) cell lines has created strong interest in applying available tools for gene transfer in murine cells to human systems. Here we present a method for the transduction of hES cells with ecotropic retroviral vectors. hES cells were transiently transfected with a construct carrying the murine retrovirus receptor mCAT1. Subsequently, the cells were exposed to replication-deficient Moloney murine leukemia virus (MoMuLV) derivatives or pseudotyped lentiviral vectors. With oncoretroviral vectors, this procedure yields overall transduction efficiencies of up to 20% and permits selection of permanently transduced clones with high frequency. Selected clones maintained expression of pluripotency-associated markers and exhibited multi-germ layer differentiation both in vitro and in vivo. HES cell-derived somatic cells including neural progeny maintained high levels of transgene expression. Lentiviral vectors pseudotyped with the MoMuLV envelope could be introduced in the same manner with efficiencies of up to 33%. Transgene expression of lentivirally transduced hES cells remained permanent after differentiation even without selection pressure. Bypassing the regulatory issues associated with the use of amphotropic retroviral systems and exploiting the large pool of existing murine vectors, this method provides a safe and versatile tool for gene transfer and lineage analysis in hES cells and their progeny.

Animals↗
PubMed2004

Hematopoietic stem cells convert into liver cells within days without fusion.

Both plasticity and cell fusion have been suggested to have a role in germ-layer switching. To understand the mechanisms underlying cell fate changes, we have examined a highly enriched population of hematopoietic stem cells (HSCs) in vitro or in vivo in response to injury for liver-specific phenotypic and functional changes. Here we show that HSCs become liver cells when cocultured with injured liver separated by a barrier. Chromosomal analyses and tissue-specific gene and/or protein expression show that microenvironmental cues rather than fusion are responsible for conversion in vitro. We transplanted HSCs into liver-injured mice and observed that HSCs convert into viable hepatocytes with increasing injury. Notably, liver function was restored 2-7 d after transplantation. We conclude that HSCs contribute to the regeneration of injured liver by converting into functional hepatocytes without fusion.

Animals↗
PubMed2005

POPK-1/Sad-1 kinase is required for the proper translocation of maternal mRNAs and putative germ plasm at the posterior pole of the ascidian embryo.

Maternal mRNAs localized to specific regions in eggs play important roles in the establishment of embryonic axes and germ layers in various species. Type I postplasmic/PEM mRNAs, which are localized to the posterior-vegetal cortex (PVC) of fertilized ascidian eggs, such as the muscle determinant macho-1 mRNA, play key roles in embryonic development. In the present study, we analyzed the function of the postplasmic/PEM RNA Hr-POPK-1, which encodes a kinase of Halocynthia roretzi. When the function of POPK-1 was suppressed by morpholino antisense oligonucleotides, the resulting malformed larvae did not form muscle or mesenchyme, as in macho-1-deficient embryos. Epistatic analysis indicated that POPK-1 acts upstream of macho-1. When POPK-1 was knocked down, localization of every Type I postplasmic/PEM mRNA examined, including macho-1, was perturbed, showing diffuse early distribution and eventual concentration into a smaller area. This is the probable reason for the macho-1 dysfunction. The postplasmic/PEM mRNAs such as macho-1 and Hr-PEM1 are co-localized with the cortical endoplasmic reticulum (cER) and move with it after fertilization. Eventually they become highly concentrated into a subcellular structure, the centrosome-attracting body (CAB), at the posterior pole of the cleaving embryos. The suppression of POPK-1 function reduced the size of the domain of concentrated cER at the posterior pole, indicating that POPK-1 is involved in the movement of postplasmic/PEM RNAs via relocalization of cER. The CAB also shrank. These results suggest that Hr-POPK-1 plays roles in concentration and positioning of the cER, as well as in the concentration of CAB materials, such as putative germ plasm, in the posterior blastomeres.

Animals↗
PubMed1993

Anterior mesendoderm induces mouse Engrailed genes in explant cultures.

We have developed germ layer explant culture assays to study the role of mesoderm in anterior-posterior (A-P) patterning of the mouse neural plate. Using isolated explants of ectodermal tissue alone, we have demonstrated that the expression of Engrailed-1 (En-1) and En-2 genes in ectoderm is independent of mesoderm by the mid- to late streak stage, at least 12 hours before their onset of expression in the neural tube in vivo at the early somite stage. In recombination explants, anterior mesendoderm from headfold stage embryos induces the expression of En-1 and En-2 in pre- to early streak ectoderm and in posterior ectoderm from headfold stage embryos. In contrast, posterior mesendoderm from embryos of the same stage does not induce En genes in pre- to early streak ectoderm but is able to induce expression of a general neural marker, neurofilament 160 x 10(3) M(r). These results provide the first direct evidence for a role of mesendoderm in induction and regionalization of neural tissue in mouse.

Animals↗