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Results for “germ layer specification”

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At least 37 records · Page 2Linked to original sources
PubMed2004

Investigating the origins of triploblasty: 'mesodermal' gene expression in a diploblastic animal, the sea anemone Nematostella vectensis (phylum, Cnidaria; class, Anthozoa).

Mesoderm played a crucial role in the radiation of the triploblastic Bilateria, permitting the evolution of larger and more complex body plans than in the diploblastic, non-bilaterian animals. The sea anemone Nematostella is a non-bilaterian animal, a member of the phylum Cnidaria. The phylum Cnidaria (sea anemones, corals, hydras and jellyfish) is the likely sister group of the triploblastic Bilateria. Cnidarians are generally regarded as diploblastic animals, possessing endoderm and ectoderm, but lacking mesoderm. To investigate the origin of triploblasty, we studied the developmental expression of seven genes from Nematostella whose bilaterian homologs are implicated in mesodermal specification and the differentiation of mesodermal cell types (twist, snailA, snailB, forkhead, mef2, a GATA transcription factor and a LIM transcription factor). Except for mef2, the expression of these genes is largely restricted to the endodermal layer, the gastrodermis. mef2 is restricted to the ectoderm. The temporal and spatial expression of these 'mesoderm' genes suggests that they may play a role in germ layer specification. Furthermore, the predominantly endodermal expression of these genes reinforces the hypothesis that the mesoderm and endoderm of triploblastic animals could be derived from the endoderm of a diploblastic ancestor. Alternatively, we consider the possibility that the diploblastic condition of cnidarians is a secondary simplification, derived from an ancestral condition of triploblasty.

Amino Acid Sequence↗
PubMed1998

The role of maternal VegT in establishing the primary germ layers in Xenopus embryos.

VegT is a T-box transcription factor whose mRNA is synthesized during oogenesis and localized in the vegetal hemisphere of the egg and early embryo. We show that maternally expressed VegT controls the pattern of primary germ layer specification in Xenopus embryos. Reduction of the maternal store completely alters the fates of different regions of the blastula so that animal cell fate is changed from epidermis and nervous system to epidermis only, equatorial cell fate is changed from mesoderm to ectoderm, and vegetal cell fate is changed from endoderm to mesoderm and ectoderm. Vegetal cells lose their capacity both to form endoderm and to release mesoderm-inducing signals. These results show that a single maternally expressed gene controls the patterning of the Xenopus blastula.

Animals↗
PubMed2001

Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types.

The controlled differentiation of pluripotent cells will be a prerequisite for many cell therapies. We have previously reported homogeneous conversion of embryonic stem (ES) cells in vitro to early primitive ectoderm-like (EPL) cells, equivalent to early primitive ectoderm, an obligatory differentiation intermediate between ES cells and somatic cell populations. Early primitive ectoderm-like cells differentiated within aggregates form mesodermal lineages at the expense of ectoderm. In this work we demonstrate that the failure of EPL cells to form ectodermal cell types does not reflect an inherent restriction in developmental potential. Early primitive ectoderm-like cells form ectodermal derivatives such as neurons in response to neural inducers such as retinoic acid, or when differentiated in the environment provided by ES cell embryoid bodies. This could be explained by signals from the extraembryonic cell type visceral endoderm which forms in differentiating ES cell but not EPL cell aggregates. Consistent with this possibility, culture of EPL cell aggregates in the presence of visceral endoderm-like signals did not prevent differentiation of the pluripotent cells, but resulted in suppression of mesoderm formation. These results suggest a role for visceral endoderm in regulation of germ layer specification from pluripotent cells, and can be integrated into a model for cell differentiation in vitro and in vivo.

Blotting, Northern↗
PubMed2004

The cnidarian and the canon: the role of Wnt/beta-catenin signaling in the evolution of metazoan embryos.

In a recent publication, Wikramanayake and colleagues have implicated the canonical Wnt/beta-catenin signaling pathway as a mediator of axial polarity and germ-layer specification in embryos of the cnidarian Nematostella. In this anthozoan, beta-catenin is localized in nuclei of blastomeres in one region of the 16- to 32-cell embryo whose descendants subsequently form the entoderm of the embryo. They claim that the pattern of nuclear localization is significant for two reasons: (1) when nuclear localization of beta-catenin was inhibited, gastrulation does not occur, and (2) when localization of beta-catenin took place in all cells of the pregastrula embryo, the number of entodermal cells increases. Since the Wnt/beta-catenin signaling pathway also plays a role in establishing axial polarity and specifying endoderm and mesoderm in a number of bilaterians, Wikramanayake et al. imply that this developmental mechanism is an evolutionary inheritance from a radially symmetrical ancestor. Some of the gaps in the current evidence, which must be filled to evaluate their interpretation, are discussed.

Animals↗
PubMed1990

Regulation of the segmentation gene fushi tarazu has been functionally conserved in Drosophila.

An evolutionary approach was applied to identify elements involved in the regulation of the segmentation gene fushi tarazu (ftz) by comparing the Drosophila melanogaster ftz gene with its Drosophila hydei homologue. The overall organization of the ftz gene is very similar in both species. Surprisingly, ftz proved to be inverted in the ANT-C of D. hydei with respect to D. melanogaster. Strong homologies extend over the entire 6 kb of the ftz upstream region with the best match in the 'upstream element'. We identified several highly conserved boxes embedded in unrelated sequences that correspond extremely well to two germ layer specific enhancers in the upstream element. Transformation experiments revealed that D. hydei ftz gene products can restore D. melanogaster ftz function and, furthermore, that trans-acting factors from D. melanogaster recognize and control D. hydei ftz regulatory elements. These findings indicate a conservation of the entire regulatory network among segmentation genes for several millions of years during the evolution of Drosophila.

Animals↗
PubMed2005

Status of RNAs, localized in Xenopus laevis oocytes, in the frogs Rana pipiens and Eleutherodactylus coqui.

Early development in the frog model, Xenopus laevis, is governed by RNAs, localized to the vegetal cortex of the oocyte. These RNAs include Xdazl RNA, which is involved in primordial germ cell formation, and VegT RNA, which specifies the mesoderm and endoderm. In order to determine whether orthologues of these RNAs are localized and have similar functions in other frogs, we cloned RpDazl and RpVegT from Rana pipiens, a frog that is phylogenetically distant from X. laevis. RNAs from both genes are localized to the vegetal cortex of the R. pipiens oocyte, indicating that the vegetal localization is likely the basal state. The animal location of EcVegT RNA in Eleutherodactylus coqui that we found previously (Beckham et al., 2003) is then a derived state, probably due to the great increase in egg size required for direct development of this species. To answer the question of function, we injected RpVegT or EcVegT RNAs into X. laevis embryos, and assayed animal caps for gene expression. Both of these RNAs induced the expression of endodermal, mesodermal, and organizer genes, showing that the function of RpVegT and EcVegT as meso-endodermal determinants is conserved in frogs. The RNA localizations and the function of VegT orthologues in germ layer specification may be synapomorphies for anuran amphibians.

Amino Acid Sequence↗
PubMed2008

Maternal control of pattern formation in Xenopus laevis.

We review the essential role of maternal factors in pattern formation for Xenopus laevis, focusing on VegT, Vg1, and Wnt11. Results from loss of function experiments demonstrate a clear requirement for these genes in germ layer specification, dorsal-ventral axis formation, and convergence extension. We also discuss these genes in the broader context of metazoan development, exploring whether and how their functions in the X. laevis model organism may or may not be conserved in other species. Wnt11 signaling in particular provides a classic example where understanding context in development is crucial to understanding function. Genomic sequencing, gene expression, and functional screening data that are becoming available in more species are providing invaluable aid to decoding and modeling signaling pathways. More work is needed to develop a comprehensive catalog of the Wnt signaling, T-box, and TGF-beta genes in metazoans both near and far in evolutionary distance. We finally discuss some specific experimental and modeling efforts that will be needed to understand the behavior of these signaling networks in vivo so that we can interpret these critical pathways in an evolutionary framework.

Animals↗
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↗
PubMed2003

Regulation of nodal and BMP signaling by tomoregulin-1 (X7365) through novel mechanisms.

During early vertebrate development, members of the transforming growth factor beta (TGFbeta) family play important roles in a variety of processes, including germ layer specification, patterning, cell differentiation, migration, and organogenesis. The activities of TGFbetas need to be tightly controlled to ensure their function at the right time and place. Despite identification of multiple regulators of Bone Morphogenetic Protein (BMP) subfamily ligands, modulators of the activin/nodal class of TGFbeta ligands are limited, and include follistatin, Cerberus, and Lefty. Recently, a membrane protein, tomoregulin-1 (TMEFF1, originally named X7365), was isolated and found to contain two follistatin modules in addition to an Epidermal Growth Factor (EGF) domain, suggesting that TMEFF1 may participate in regulation of TGFbeta function. Here, we show that, unlike follistatin and follistatin-related gene (FLRG), TMEFF1 inhibits nodal but not activin in Xenopus. Interestingly, both the follistatin modules and the EGF motif contribute to nodal inhibition. A soluble protein containing the follistatin and the EGF domains, however, is not sufficient for nodal inhibition; the location of TMEFF1 at the membrane is essential for its function. These results suggest that TMEFF1 inhibits nodal through a novel mechanism. TMEFF1 also blocks mesodermal, but not epidermal induction by BMP2. Unlike nodal inhibition, regulation of BMP activities by TMEFF1 requires the latter's cytoplasmic tail, while deletion of either the follistatin modules or the EGF motif does not interfere with the BMP inhibitory function of TMEFF1. These results imply that TMEFF1 may employ different mechanisms in the regulation of nodal and BMP signals. In Xenopus, TMEFF1 is expressed from midgastrula stages onward and is enriched in neural tissue derivatives. This expression pattern suggests that TMEFF1 may modulate nodal and BMP activities during neural patterning. In summary, our data demonstrate that tomoregulin-1 is a novel regulator of nodal and BMP signaling during early vertebrate embryogenesis.

Activins↗
PubMed2003

Characterization and differentiation of human embryonic stem cells.

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.

Animals↗
PubMed2005

Establishment of a chick embryo model for analyzing liver development and a search for candidate genes.

The liver plays a crucial role in metabolism. There is considerable interest in how the liver develops, as such knowledge could prove of importance in regenerative medicine. However, our understanding of liver development remains somewhat limited. We have developed a model system using the chick embryo that is cost effective and is easy to manipulate experimentally. We performed four fundamental studies: (i) construction of an atlas of the developing chick liver; (ii) identification of differentiation marker genes in the developing chick embryo; (iii) development of germ-layer specific electroporation; and (iv) establishment of organ culture from the developing chick liver. Using this system, we have been able to demonstrate the functions of candidate genes within a shorter period and in a more cost-effective manner. In parallel with the establishment of this system, we examined the expression patterns of genes known to be required for organ development in the developing chick embryo in order to identify genes also involved in liver development. To date, we have found sixteen genes that are expressed in the developing chick liver (GELD, genes expressed in liver development). This knowledge will be fundamental to the establishment of the basic technology for engineering liver tissue in the future.

Animals↗
PubMed2005

Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development.

To date, hematopoietic development of human embryonic stem cells (hESCs) has been limited to cell lines cultured in the presence of either mouse embryonic fibroblasts (MEFs) or MEF-conditioned media (MEF-CM). Anonymous xenogenic factors from MEFs or MEF-CM complicate studies of hESC self-renewal and also raise concerns for the potential clinical applications of generating primitive hematopoietic cells from hESC lines maintained under these ambiguous conditions. Here, we demonstrate that hESCs can be cultured over 30 passages in defined conditions in the absence of MEFs or MEF-CM using only serum replacement (SR) media and high concentrations of basic fibroblast growth factor (SR-bFGF). Similar to hESCs cultured in MEF-CM, hESCs cultured in SR-bFGF sustained characteristics of undifferentiated hESCs, proliferative potential, normal karyotype, in vitro and in vivo 3 germ-layer specification and gave rise to hemogenic-endothelial precursors required for subsequent primitive hematopoietic development. Our report demonstrates that anonymous factors produced by feeder cells are not necessary for hESC maintenance and subsequent hematopoietic specification, thereby providing a defined system for studies of hESC self-renewal and hESC-derived hematopoiesis.

Alkaline Phosphatase↗
PubMed2006

Conserved roles for Oct4 homologues in maintaining multipotency during early vertebrate development.

All vertebrate embryos have multipotent cells until gastrulation but, to date, derivation of embryonic stem (ES) cell lines has been achieved only for mouse and primates. ES cells are derived from mammalian inner cell mass (ICM) tissue that express the Class V POU domain (PouV) protein Oct4. Loss of Oct4 in mice results in a failure to maintain ICM and consequently an inability to derive ES cells. Here, we show that Oct4 homologues also function in early amphibian development where they act as suppressors of commitment during germ layer specification. Antisense morpholino mediated PouV knockdown in Xenopus embryos resulted in severe posterior truncations and anterior neural defects. Gastrulation stage embryos showed reduced expression of genes associated with uncommitted marginal zone cells, while the expression of markers associated with more mature cell states was expanded. Importantly, we have tested PouV proteins from a number of vertebrate species for the ability to substitute Oct4 in mouse ES cells. PouV domain proteins from both Xenopus and axolotl could support murine ES cell self-renewal but the only identified zebrafish protein in this family could not. Moreover, we found that PouV proteins regulated similar genes in ES cells and Xenopus embryos, and that PouV proteins capable of supporting ES cell self-renewal could also rescue the Xenopus PouV knockdown phenotype. We conclude that the unique ability of Oct4 to maintain ES cell pluripotency is derived from an ancestral function of this class of proteins to maintain multipotency.

Amino Acid Sequence↗
PubMed1994

Sp1/egr-like zinc-finger protein required for endoderm specification and germ-layer formation in Drosophila.

Much of our present knowledge of the biological processes involved in pattern formation in Drosophila is derived from segmentation analysis. Comparatively little is known about the genetic requirement and mechanisms underlying the formation and separation of germ layers by morphogenetic movements during gastrulation. Here we show that the Drosophila gene huckebein (hkb), a member of the gap-gene class of segmentation genes, is required for germ-layer formation at blastoderm. Absence of the hkb product, an Sp1/egr-like zinc-finger protein, causes the ectodermal and mesodermal primordia to expand at the expense of endoderm anlagen. Conversely, ectopic expression of hkb inhibits the formation of the major gastrulation fold which gives rise to the mesoderm and prevents normal segmentation in the ectoderm. Thus, hkb is necessary for endoderm development and its activity defines spatial limits within the blastoderm embryo in which the germ layers are established.

Amino Acid Sequence↗
PubMed2002

The expression of the imprinted gene Ipl is restricted to extra-embryonic tissues and embryonic lateral mesoderm during early mouse development.

Genes with restricted expression within the developing embryo represent valuable tools as they allow distinct tissue types to be distinguished and studied. In order to identify genes that are expressed within a particular germ layer, a differential screen was performed using germ layer-specific cDNA libraries derived from gastrulation stage mouse embryos. The gene expression profiles of the germ layers were compared following the hybridisation of some 20,000 cDNA clones with probes derived from germ layer-specific Ectoderm, Mesoderm and Endoderm libraries. A cDNA clone (50c15) was identified that hybridised with the Mesoderm-derived probe but not Ectoderm or Endoderm. 50c15 derives from Ipl/Tssc3/BWR1C, an imprinted gene which in human maps to chromosome 11p15.5. This region has been associated with Beckwith-Weidemann Syndrome, Wilms' tumour and ovarian, breast and lung cancer. In the gastrulating mouse embryo, wholemount RNA in situ hybridisation revealed that Ipl expression is restricted not only to the mesodermal germ layer, but specifically to lateral mesoderm and the most posterior extent of the primitive streak from which lateral and extra-embryonic mesoderm is derived. Moreover, Ipl is expressed in extra-embryonic tissues prior to gastrulation and afterwards in extra-embryonic mesoderm, ectoderm and endoderm. This expression profile indicates that Ipl is a good molecular marker for embryonic mesoderm and extra-embryonic tissues. In addition heterotopic grafting studies indicate that nascent mesoderm, which expresses Ipl, is restricted in its potential and therefore may be committed to its fate.

Animals↗
PubMed2005

Measuring cell adhesion forces of primary gastrulating cells from zebrafish using atomic force microscopy.

During vertebrate gastrulation, progenitor cells of different germ layers acquire specific adhesive properties that contribute to germ layer formation and separation. Wnt signals have been suggested to function in this process by modulating the different levels of adhesion between the germ layers, however, direct evidence for this is still lacking. Here we show that Wnt11, a key signal regulating gastrulation movements, is needed for the adhesion of zebrafish mesendodermal progenitor cells to fibronectin, an abundant extracellular matrix component during gastrulation. To measure this effect, we developed an assay to quantify the adhesion of single zebrafish primary mesendodermal progenitors using atomic-force microscopy (AFM). We observed significant differences in detachment force and work between cultured mesendodermal progenitors from wild-type embryos and from slb/wnt11 mutant embryos, which carry a loss-of-function mutation in the wnt11 gene, when tested on fibronectin-coated substrates. These differences were probably due to reduced adhesion to the fibronectin substrate as neither the overall cell morphology nor the cell elasticity grossly differed between wild-type and mutant cells. Furthermore, in the presence of inhibitors of fibronectin-integrin binding, such as RGD peptides, the adhesion force and work were strongly decreased, indicating that integrins are involved in the binding of mesendodermal progenitors in our assay. These findings demonstrate that AFM can be used to quantitatively determine the substrate-adhesion of cultured primary gastrulating cells and provide insight into the role of Wnt11 signalling in modulating cell adhesion at the single cell scale.

Animals↗
PubMedDate not supplied

Neural transdifferentiation of mesenchymal stem cells--a critical review.

The classic concept of stem cell differentiation can be illustrated as driving into a series of one-way streets, where a given stem cell through generations of daughter cells becomes correspondingly restricted and committed towards a definitive lineage with fully differentiated cells as end points. According to this concept, tissue-derived adult stem cells can only give rise to cells and cell lineages found in the natural, specified tissue of residence. During the last few years it has, however, been reported that under certain experimental conditions adult stem cells may lose their tissue or germ layer-specific phenotypes and become reprogrammed to transdifferentiate into cells of other germ layers and tissues. The transdifferentiation process is referred to as "stem cell plasticity". Mesenchymal stem cells, present in various tissues, including bone marrow, have--besides differentiation into bone, cartilage, smooth muscle and skeletal muscle--also been reported to transdifferentiate into skin, liver and brain cells (neurons and glia). Conversely, neural stem cells have been reported to give rise to blood cells. The actual occurrence of transdifferentiation is currently much debated, but would have immense clinical potential in cell replacement therapy and regenerative medicine. Controlled neural differentiation of human mesenchymal stem cells might thus become an important source of cells for cell therapy of neurodegenerative diseases, since autologous adult mesenchymal stem cells are more easily harvested and effectively expanded than corresponding neural stem cells. This article provides a critical review of the reports of neural transdifferentiation of mesenchymal stem cells, and proposes a set of criteria to be fulfilled for validation of transdifferentiation.

Animals↗
PubMed2025

Foxh1 is a locus-specific PRC2 recruiter governing germ layer silencing.

Polycomb Repressive Complex 2 (PRC2) establishes H3K27me3 marks to shape spatiotemporal gene expression during embryogenesis. While its dysregulation is linked to developmental disorders, cancer, and aging, the mechanisms guiding PRC2 to specific genomic loci remain a subject of ongoing debate. A prevailing model proposes that PRC2 recruitment occurs via its intrinsic affinity for chromatin rather than through sequence-specific transcription factors. Here, we provide evidence that the maternally deposited pioneer transcription factor Foxh1 plays a critical role in directing PRC2 to specific genomic loci during zygotic genome activation in Xenopus. Foxh1 is a critical transcription factor mediating Nodal signaling, but it also plays an earlier role by pre-binding enhancers prior to signaling activation. This pre-binding is essential for forming enhanceosome complexes that trigger mesendodermal gene expression and drive gastrulation, in cooperation with other maternal transcription factors. Using maternal Foxh1-null embryos, we demonstrate that Foxh1 directly recruits Ezh2, the catalytic subunit of PRC2, to Foxh1-bound loci. Loss of Foxh1 impairs Ezh2 recruitment, leading to a global reduction in H3K27me3. These findings support a dual-function model in which Foxh1 not only activates endodermal gene expression in endoderm, but also recruits PRC2 to silence the same genes in ectoderm. This dual activity of Foxh1 allows the spatially coordinated epigenetic states of the endodermal gene regulatory program during early embryogenesis.

CRISPR/Cas9↗