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PubMed2006

A WNT of things to come: evolution of Wnt signaling and polarity in cnidarians.

The conserved family of Wnt signaling molecules mediates various developmental processes including governing cell fate, proliferation, and polarity. The diverse developmental functions of the Wnt genes in bilaterians have obscured the evolutionary origin of this important signaling pathway. Recent work in the Cnidaria has shown the diversity of Wnt genes, and regulatory components of Wnt signaling, evolved early in metazoan evolution, prior to the divergence of cnidarians and bilaterians. Evidence from Hydra and the sea anemone, Nematostella, demonstrates a role for Wnt signaling in axis formation and patterning, as well as gastrulation and germ-layer specification. In this review, we examine what is currently known about Wnt signaling in cnidarians, and discuss what this group of "simple" animals may reveal about the evolution of Wnt signaling and polarity.

Animals↗
PubMed2004

Gastrulation dynamics: cells move into focus.

During vertebrate gastrulation, a relatively limited number of blastodermal cells undergoes a stereotypical set of cellular movements that leads to formation of the three germ layers: ectoderm, mesoderm and endoderm. Gastrulation, therefore, provides a unique developmental system in which to study cell movements in vivo in a fairly simple cellular context. Recent advances have been made in elucidating the cellular and molecular mechanisms that underlie cell movements during zebrafish gastrulation. These findings can be compared with observations made in other model systems to identify potential general mechanisms of cell migration during development.

Animals↗
PubMed2004

Tracking mesoderm formation and specification to the hemangioblast in vitro.

During development of the mouse embryo, blood cells are generated from mesodermal precursors at specific times and locations. Using various in vivo and in vitro systems, we are now starting to understand the cascade of molecular events leading to the commitment of mesoderm and the formation of the first blood precursors, the hemangioblast. The in vitro differentiation of embryonic stem (ES) cells has proved to be an invaluable model to study lineage commitment, because one can access and easily manipulate large quantities of early progenitor cells. To help us track mesodermal subpopulations and study their specification toward blood lineages, we have engineered an ES cell line that expresses the green fluorescence protein exclusively in the mesoderm germ layer, under the control of the Brachyury regulatory sequences.

Animals↗
PubMed2006

Definitive hematopoiesis from endothelial cells in the mouse embryo; a simple guide.

Circulation is composed of two interactive systems, the cardiovascular and the hematopoietic, which affect each other. Recently, endothelial progenitor cells/angioblasts have been identified in the circulation of the adult mouse and human. Furthermore, some hematopoietic cells (HCs) have been shown to contribute to angiogenesis, suggesting that HCs can transdifferentiate into endothelial cells (ECs). Although these concepts in adult are still controversial, understanding the mechanisms of the relationship between ECs and HCs would benefit the clinical application for cardiovascular and hematologic disorders. Both ECs and HCs are considered to be derived from a common germ layer, the mesoderm, and have more intimate relationship in embryo than in adult. Here, we describe the relationship between ECs and HCs with special attention to the hemogenic ECs in the mouse embryo.

Adult↗
PubMed2004

Monitoring of teratogenic effects in vitro by analysing a selected gene expression pattern.

The development of in vitro methods for regulatory embryotoxicity testing is challenging since the understanding of chemical effects on the mammalian development is still poor. The aim of the project is to identify marker genes during in vitro cell differentiation of murine embryonic stem cells, in order to predict chemical effects on cell differentiation of specific target tissues. The present study is focusing on the expression pattern by using semi-quantitative reverse transcriptase (RT)-PCR of key genes involved in cardiomyocytes development; i.e. Oct-4, Brachyury, Nkx2.5 and alpha myosin heavy chain (alpha-MHC). Two reference chemicals with well-known in vivo data have been analysed by using this approach: retinoic acid and lithium chloride. Retinoic acid has been selected as a teratogen affecting several target tissues, whereas lithium chloride has been described to affect the development of the cardiovascular system. We demonstrate that retinoic acid already affects in the early stage of germ layer formation, which was demonstrated by a change of Oct-4 and Brachyury gene expression. As we expected, the expression of cardiac specific genes (Nkx2.5, alpha-MHC) has been also modified. In contrary, the Oct-4 and Brachyury expression was not changed by lithium treatment. In this case, we observed a modification in the normal gene expression pattern, for alpha-MHC and Nkx2.5, demonstrating that lithium chloride affects the later stage of heart development. These data suggest that the inclusion of selective target organ genes in an established embryotoxicity test allows to predict effects of chemicals and drugs to the heart development.

Animals↗
PubMed2005

The transplantation of neural stem cells and predictive factors in hematopoietic recovery in irradiated mice.

A number of surprising observations have shown that stem cells, in suitable conditions, have the ability to produce a whole spectrum of cell types, regardless, whether these tissues are derived from the same germ layer or not. This phenomenon is called stem cell plasticity, which means that tissue-specific stem cells are mutually interchangeable. In our experiments, as a model, we used neural stem cells (NSCs) harvested from fetal (E14-15) neocortex and beta-galactosidase positive. In the first experiment we found that on days 12 and 30 after sub-lethal irradiation (LD 8.5 Gy) and (beta-galactosidase(+)) NSCs transplantation all mice survived, just as the group with bone marrow transplantation. Moreover, the bone marrow of mice transplanted NSCs contained the number of CFU-GM colonies with beta-galactosidase(+) cells which was as much as 50% higher. These differences were statistically significant, p<0.001. In the second experiment, we studied kinetics of (beta-galactosidase(+)) NSCs after their transplantation to sub-lethally irradiated mice. Histochemistry of tissues was performed on days 12 and 30 post-transplantation, and beta-galactosidase(+) cells were detected with the help of histochemical examination of removed tissues (lung, liver, spleen, thymus, and skeletal muscle). In tissues removed on day 12 post-transplantation, we found a significantly higher number of beta-galactosidase(+) cells in the spleen and thymus on day 30. While we presumed the presence beta-galactosidase(+) cells in the spleen, as spleen and reticuloendothelial system represent an important retaining system for different cell types, the presence of beta-galactosidase(+) cells in the thymus was rather surprising but very interesting. This indicates a certain mutual and close interconnection of transplanted stem cells and immune system in an adult organism. In the third experiment, we verified the mutual interchange of Sca-1 surface antigen in the bone marrow cells and NSCs before transplantation. Analysis of this antigen showed 24.8% Sca-1 positive cells among the bone marrow cells, while NSCs were Sca-1 negative. Our experiments show that NSCs share hemopoietic identity and may significantly influence the recovery of damaged hematopoiesis but do not have typical superficial markers as HSCs. This result is important for the determination of predictive factors for hemopoiesis recovery, for stem cell plasticity and for their use in the cell therapy.

Animals↗
PubMedDate not supplied

Green fluorescent proteins in receptor research: an emerging tool for drug discovery.

In the last five years, green fluorescent protein (GFP) has emerged from being a mere curiosity to become a reliable tool for molecular pharmacological research. GFP produces an intense and stable green fluorescence noncatalytically by absorbing blue light maximally at 395 nm and emitting green light with a peak at 509 nm. It consists of 238 amino acids and its molecular mass is 27-30 kDa. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence to be utilised in nonnative organisms, wherein it can be used as a reporter. This use of GFP permits real-time analysis of receptor dynamics. The emitted fluorescence can be used as a nontoxic marker and detected using fluorescence-activated cell sorting (FACS), thus avoiding any staining procedure, expensive mRNA analysis or hazardous radiolabeled binding assays. The potential value of GFP has also been recognized in orphan receptor research, where various GFP-tagged therapeutic proteins have been constructed in an attempt to identify the endogenous ligand(s). These chimeric proteins have been used to determine the site and time course of receptor expression and to relate receptor dynamics with therapeutic outcome. The preparation of new GFP constructs for identifying germ layer cells (endodermal, ectodermal, and mesodermal), as well as neuronal, haematopoietic, endothelial, and cartilage cells, has provided a useful battery of tissue/receptor-specific screening assays for new chemical entities. Genetically engineered cells with GFP expression have provided a valuable tool for automated analysis, and can be adapted for high-throughput systems. GFP is being increasingly utilised for the study of receptor dynamics, where, having already proved beneficial, it will likely continue to contribute towards the search for new classes of drugs, as well as to "de-orphaning" orphan receptors.

Animals↗
PubMed2004

Zebrafish mnx genes in endocrine and exocrine pancreas formation.

The pancreas consists of two components, which exert distinct homeostatic function, an endocrine part that secretes hormones including insulin and an exocrine part that produces digestive enzymes. In mouse, one of the factors essential for development of the pancreas is the Mnx-class homeobox transcription factor Hb9. Genetic studies showed that Hb9 is required for both initial morphogenesis of the pancreas as well as subsequent differentiation of insulin-producing beta-cells [Nat. Genet. 23 (1999) 71; Nat. Genet. 23 (1999) 67]. To get a better understanding of what role mnx genes play in pancreas development, we isolated and characterized mnx genes in the model organism zebrafish. We found one gene with homology to hb9 orthologs and two that display homology to the related chicken mnr2. Embryonic expression of the zebrafish mnx genes is very dynamic and is detected in derivatives of all three germ layers. Endodermal expression of hb9 takes place in the early gut endoderm and, later, in the endocrine pancreas and the swim bladder. In addition, one of the mnr2 genes, mnr2a, shows expression in an endodermal cell population that is initially intermingled with insulin-positive cells and that later becomes restricted to the exocrine pancreas. In knockdown studies using antisense morpholinos, we show that hb9 is essential for differentiation of the insulin-producing beta-cells but unlike mouse Hb9 is not needed for early morphogenesis of the pancreas. In contrast, mnr2a is required during late morphogenesis of the exocrine pancreas. In summary, our data suggest a tissue-specific mnx-expression code in the zebrafish pancreas and they reveal a novel role of an mnr2-related gene.

Air Sacs↗
PubMed2004

Differences between human and mouse embryonic stem cells.

We compared gene expression profiles of mouse and human ES cells by immunocytochemistry, RT-PCR, and membrane-based focused cDNA array analysis. Several markers that in concert could distinguish undifferentiated ES cells from their differentiated progeny were identified. These included known markers such as SSEA antigens, OCT3/4, SOX-2, REX-1 and TERT, as well as additional markers such as UTF-1, TRF1, TRF2, connexin43, and connexin45, FGFR-4, ABCG-2, and Glut-1. A set of negative markers that confirm the absence of differentiation was also developed. These include genes characteristic of trophoectoderm, markers of germ layers, and of more specialized progenitor cells. While the expression of many of the markers was similar in mouse and human cells, significant differences were found in the expression of vimentin, beta-III tubulin, alpha-fetoprotein, eomesodermin, HEB, ARNT, and FoxD3 as well as in the expression of the LIF receptor complex LIFR/IL6ST (gp130). Profound differences in cell cycle regulation, control of apoptosis, and cytokine expression were uncovered using focused microarrays. The profile of gene expression observed in H1 cells was similar to that of two other human ES cell lines tested (line I-6 and clonal line-H9.2) and to feeder-free subclones of H1, H7, and H9, indicating that the observed differences between human and mouse ES cells were species-specific rather than arising from differences in culture conditions.

Animals↗
PubMed2004

Analysis of forkhead and snail expression reveals epithelial-mesenchymal transitions during embryonic and larval development of Nematostella vectensis.

The winged helix transcription factor Forkhead and the zinc finger transcription factor Snail are crucially involved in germ layer formation in Bilateria. Here, we isolated and characterized a homolog of forkhead/HNF3 (FoxA/group 1) and of snail from a diploblast, the sea anemone Nematostella vectensis. We show that Nematostella forkhead expression starts during late Blastula stage in a ring of cells that demarcate the blastopore margin during early gastrulation, thereby marking the boundary between ectodermal and endodermal tissue. snail, by contrast, is expressed in a complementary pattern in the center of forkhead-expressing cells marking the presumptive endodermal cells fated to ingress during gastrulation. In a significant portion of early gastrulating embryos, forkhead is expressed asymmetrically around the blastopore. While snail-expressing cells form the endodermal cell mass, forkhead marks the pharynx anlage throughout embryonic and larval development. In the primary polyp, forkhead remains expressed in the pharynx. The detailed analysis of forkhead and snail expression during Nematostella embryonic and larval development further suggests that endoderm formation results from epithelial invagination, mesenchymal immigration, and reorganization of the endodermal epithelial layer, that is, by epithelial-mesenchymal transitions (EMT) in combination with extensive morphogenetic movements. snail also governs EMT at different processes during embryonic development in Bilateria. Our data indicate that the function of snail in Diploblasts is to regulate motility and cell adhesion, supporting that the triggering of changes in cell behavior is the ancestral role of snail in Metazoa.

Amino Acid Sequence↗
PubMed2005

The ERRalpha orphan nuclear receptor controls morphogenetic movements during zebrafish gastrulation.

Gastrulation is a process involving cellular commitment and movements whereby the three fundamental germ layers are established in vertebrates embryos. Estrogen Receptor-Related (ERR) alpha is a nuclear receptor displaying high sequence identity to the Estrogen Receptors (ERs). However, ERRalpha is unable to bind and to be regulated by estrogens or any natural ligand to date. Whereas recent studies have suggested roles for ERRalpha in bone and adipose tissue metabolism in the mouse, little is known about its roles during embryonic development. In zebrafish embryos, ERRalpha is expressed from the beginning of gastrulation at the margin of the blastoderm that represents the presumptive mesendoderm. Using loss of function (morpholinos or a dominant-negative version of the protein) and gain of function (mRNA injection) strategies, we show here that ERRalpha is involved in epiboly and convergent-extension (CE) processes in the zebrafish. Altogether, these results propose ERRalpha as a new regulator of morphogenetic movement during gastrulation, independently of cell fate determination.

Animals↗
PubMed2006

A Gbx homeobox gene in amphioxus: insights into ancestry of the ANTP class and evolution of the midbrain/hindbrain boundary.

In the vertebrate central nervous system (CNS), mutual antagonism between posteriorly expressed Gbx2 and anteriorly expressed Otx2 positions the midbrain/hindbrain boundary (MHB), but does not induce MHB organizer genes such as En, Pax2/5/8 and Wnt1. In the CNS of the cephalochordate amphioxus, Otx is also expressed anteriorly, but En, Pax2/5/8 and Wnt1 are not expressed near the caudal limit of Otx, raising questions about the existence of an MHB organizer in amphioxus. To investigate the evolutionary origins of the MHB, we cloned the single amphioxus Gbx gene. Fluorescence in situ hybridization showed that, as in vertebrates, amphioxus Gbx and the Hox cluster are on the same chromosome. From analysis of linked genes, we argue that during evolution a single ancestral Gbx gene duplicated fourfold in vertebrates, with subsequent loss of two duplicates. Amphioxus Gbx is expressed in all germ layers in the posterior 75% of the embryo, and in the CNS, the Gbx and Otx domains abut at the boundary between the cerebral vesicle (forebrain/midbrain) and the hindbrain. Thus, the genetic machinery to position the MHB was present in the protochordate ancestors of the vertebrates, but is insufficient for induction of organizer genes. Comparison with hemichordates suggests that anterior Otx and posterior Gbx domains were probably overlapping in the ancestral deuterostome and came to abut at the MHB early in the chordate lineage before MHB organizer properties evolved.

Amino Acid Sequence↗
PubMed2006

Overlapping expression of FoxA and Zic confers responsiveness to FGF signaling to specify notochord in ascidian embryos.

Differences in cell responsiveness to an inductive signal contribute to the emergence of a variety of tissue types during animal development. In ascidian embryos, the Fibroblast Growth Factor (FGF) signal secreted from endoderm cells induces several different tissue types, such as notochord, mesenchyme and brain, at different positions in the embryo at the 32-cell stage. We show here in Halocynthia roretzi that FoxA and Zic are required for notochord formation in cells that receive the FGF signal. We also show that these transcription factors, only when both are supplied, are able to induce ectopic expression of the brachyury gene, a notochord-specific marker, in cells of all the three germ layers in an FGF-dependent manner. These results suggest that FoxA and Zic confer notochord-specific responsiveness to FGF signaling. Further analyses including knockdown and over-expression experiments showed that combinatorial inputs from maternally supplied and zigotically activated factors lead to overlapping expression of FoxA and Zic in the presumptive notochord cells, which eventually activate the expression of the brachyury gene in cooperation with FGF signaling. Our data illustrate how a complex gene network specifies the notochord at its specific position within the embryo.

Animals↗
PubMed2006

Role of Src family kinases and N-Myc in spermatogonial stem cell proliferation.

Spermatogonial stem cells are required for the initiation of spermatogenesis and the continuous production of sperm. In addition, they can acquire pluripotency and differentiate into derivatives of the three embryonic germ layers when cultured in the appropriate conditions. Therefore, understanding the signaling pathways that lead to self-renewal or differentiation of these cells is of paramount importance for the treatment of infertility, the development of male contraceptives, the treatment of testicular cancers, and ultimately for tissue regeneration. In this report, we studied some of the signaling pathways triggered by glial cell line-derived neurotrophic factor (GDNF), a component of the spermatogonial stem cell niche produced by the somatic Sertoli cells. As model systems, we used primary cultures of mouse spermatogonial stem cells, a mouse spermatogonial stem cell line and freshly isolated testicular tubules. We report here that GDNF promotes spermatogonial stem cell proliferation through activation of members of the Src kinase family, and that these kinases exert their action through a PI3K/Akt-dependent pathway to up-regulate N-myc expression. Thus, to proliferate, spermatogonial stem cells activate mechanisms that are similar to the processes observed in brain stem cells and lung progenitors.

Animals↗
PubMed2004

Significant variations in differentiation properties between independent mouse ES cell lines cultured under defined conditions.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass (ICM)/epiblast of preimplantation embryos and are widely used in cell differentiation studies. We have previously observed differences in transcript and antigen expression following differentiation of ES cells lines in vitro. We have investigated this further by comparing the differentiation characteristics of five independently derived ES cell lines cultured and differentiated under defined conditions. Undifferentiated ES cell lines exhibited similar morphology and antigen/transcript marker expression. However, upon differentiation in monolayer culture by LIF withdrawal, only two of the lines expressed similar germ layer transcript profiles, and these were significantly altered compared to differentiation in serum-supplemented media. Neurofilament-68k was the only transcript marker common to all cell lines, however, induction of neuroectoderm lineages using 1 microM all-trans retinoic acid (RA) resulted in significant variations in cell number and morphology between the lines. Furthermore, neurons were only formed from clones of the two cell lines that exhibited similar transcript profiles, although the morphology was different between the two. We conclude that the independent ES cell lines in this study differ in their response to alterations in culture conditions in vitro, and the use of an appropriate cell line enables relatively homogeneous neuronal populations to be achieved in monolayer culture under defined conditions.

Animals↗
PubMed2006

Cell surface 5T4 antigen is transiently upregulated during early human embryonic stem cell differentiation: effect of 5T4 phenotype on neural lineage formation.

The 5T4 oncofoetal antigen is a cell surface glycoprotein that is transiently expressed during mouse ES cell differentiation and correlates with decreased pluripotency of such cells. We show that 5T4 antigen is transiently unregulated during HES4 and H1 human ES cell differentiation and its expression correlates with loss of the pluripotent markers OCT-4 and Tra-1-60 and upregulation of transcript markers associated with the three primary germ layers. To confirm that absence of cell surface 5T4 antigen represents a pluripotent hES cell phenotype, we performed mechanical transfer of either 5T4-ve or 5T4+ve HES4 colonies identified using live cell staining. 5T4-ve transfers maintained expression of OCT-4 in over 90% of resultant colonies, whereas 5T4+ve transfers exhibited significantly lower numbers of OCT-4-expressing colonies (92 +/- 1.4 vs. 2.9 +/- 2.0%). Interestingly, low cell density 5T4-ve colony transfers exhibited increased numbers of OCT-4-expressing colonies compared to large 5T4-ve transfers (92 +/- 1.4 vs. 63.2 +/- 1.9%). 5T4-ve and 5T4+ve HES4 and H1 ES cell lines expressed markers representative of neuroectoderm lineages, and we assessed the formation of neural lineages from these phenotypes in serum-containing medium and N2B27 medium. Expression of 5T4 was found to be inversely related to the yield of tyrosine-hydroxylase (TH+)-expressing neurons in N2B27 medium, with additional mesoderm and endoderm transcript markers detected. Homogeneous glial cell populations were derived from low cell density 5T4-ve colony transfers cultured in serum-containing medium, with TH+ neuronal formation inhibited in a cell-density-dependent manner. We conclude that the 5T4 antigen is a transient marker of hES cell differentiation and that 5T4 phenotype, colony seeding density and culture conditions significantly influence the maintenance of pluripotent hES cells and their differentiation to neural lineages.

Animals↗
PubMed2006

Rabbit embryonic stem cell lines derived from fertilized, parthenogenetic or somatic cell nuclear transfer embryos.

Embryonic stem cells were isolated from rabbit blastocysts derived from fertilization (conventional rbES cells), parthenogenesis (pES cells) and nuclear transfer (ntES cells), and propagated in a serum-free culture system. Rabbit ES (rbES) cells proliferated for a prolonged time in an undifferentiated state and maintained a normal karyotype. These cells grew in a monolayer with a high nuclear/cytoplasm ratio and contained a high level of alkaline phosphate activity. In addition, rbES cells expressed the pluripotent marker Oct-4, as well as EBAF2, FGF4, TDGF1, but not antigens recognized by antibodies against SSEA-1, SSEA-3, SSEA-4, TRA-1-10 and TRA-1-81. All 3 types of ES cells formed embryoid bodies and generated teratoma that contained tissue types of all three germ layers. rbES cells exhibited a high cloning efficiency, were genetically modified readily and were used as nuclear donors to generate a viable rabbit through somatic cell nuclear transfer. In combination with genetic engineering, the ES cell technology should facilitate the creation of new rabbit lines.

Animals↗
PubMed2004

Gastrointestinal adenocarcinoma arising in a mature cystic teratoma of the ovary.

BACKGROUND: Mature cystic teratoma of the ovary (MCTO) is the most common ovarian germ cell neoplasm and is usually diagnosed in early adulthood. Malignant transformation is rare, occurring in approximately 2% of all cases. Though malignant transformation can occur from any of the embryonic germ layers, the most common malignancy arising in these otherwise benign tumors is squamous cell carcinoma. CASE: We present a patient with a MCTO where malignant transformation of gastrointestinal epithelium resulted in moderately differentiated adenocarcinoma. After 3 years of follow-up, she remains free of disease. CONCLUSION: Although gastrointestinal epithelium is often found in MCTOs, adenocarcinoma arising from this cell type is uncommon. This is the third reported case of adenocarcinoma arising in gastrointestinal epithelium of a MCTO.

Adenocarcinoma↗