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Results for “germ layer specification”

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PubMed2000

Skeletal myogenic potential of human and mouse neural stem cells.

Distinct cell lineages established early in development are usually maintained throughout adulthood. Thus, adult stem cells have been thought to generate differentiated cells specific to the tissue in which they reside. This view has been challenged; for example, neural stem cells can generate cells that normally originate from a different germ layer. Here we show that acutely isolated and clonally derived neural stem cells from mice and humans could produce skeletal myotubes in vitro and in vivo, the latter following transplantation into adult animals. Myogenic conversion in vitro required direct exposure to myoblasts, and was blocked if neural cells were clustered. Thus, a community effect between neural cells may override such myogenic induction. We conclude that neural stem cells, which generate neurons, glia and blood cells, can also produce skeletal muscle cells, and can undergo various patterns of differentiation depending on exposure to appropriate epigenetic signals in mature tissues.

Age Factors↗
PubMed2003

Tissue injury and repair in allografts: novel perspectives.

PURPOSE OF REVIEW: Recent research achievements might considerably alter scientific concepts of pathways involved in tissue injury and repair. RECENT FINDINGS: Accumulating evidence for an important role of alloantibodies in acute and chronic allograft rejection led to a renewed interest in humoral kidney transplant rejection. Studies reassessing the mechanisms of antibody- and complement-mediated injury now shed new light on the pathogenic mechanisms underlying acute or chronic graft dysfunction and injury. A closer look at humoral effector mechanisms revealed that endothelial cell activation and injury may play a key role in humoral rejection, and further uncovered an important interplay between humoral and cellular alloimmunity. Regeneration of cells after injury has been thought to rely on activation of local progenitor cells. Recent investigation indicates that regeneration of grafted solid organs is not exclusively based on self-renewal of tissues but obviously also involves repopulation of the graft by recipient cells, creating chimerism in the vasculature and other compartments. Besides reparative compensation of cell loss, chimerism of endothelial cells might also alter immunologic properties of the graft, thus favoring adaptation and graft survival. On the other hand, however, myofibroblasts mediating deleterious arterial intimal proliferation may also be of recipient origin. A possible source of graft-repopulating recipient cells are bone marrow-derived adult stem cells with the amazing capacity of differentiating into cell types of all three germ cell layers. SUMMARY: Reliable diagnosis of humoral mechanisms in allograft rejection and identification of involved effector mechanisms should provide the basis for development and targeted application of specific anti-humoral treatment. Recently emerged new concepts of mechanisms underlying tissue regeneration might pave the way for entirely new therapeutic approaches in human disease.

Adaptation, Physiological↗
PubMed1998

Hydrophobic attachment of Trypanosoma cruzi to the rectal cuticle of Triatoma infestans and its influence on metacyclogenesis - a review.

Trypanosoma cruzi colonizes mainly the rectum of the vector, especially the rectal glands. We investigated the basic architecture of the rectal cuticle of Triatoma infestans and the mode of attachment of T. cruzi in the small intestine and the rectum. In addition, we determined the capacity of culture-derived epimastigotes to attach to artificial substrates and the influence of attachment on metacyclogenesis. After incubation of the rectum with wheat germ lectin (WGA) coupled to gold particles, the procuticle contained chitin, but the two layers of the epicuticle and the superficial layer bordering the rectal lumen did not. The specific fluorochrom Nile Red stained the entire rectal cuticle green, indicating the waxy composition of the superficial layer. In electron microscopic analysis the parasites were attached to the hydrophobic superficial wax layer but not to the epithelium of the midgut. In vitro culture-derived epimastigotes attached with a high affinity to all hydrophobic substrates tested, whereas hydrophilic substrates did not permit attachment. Emulsified hexadecane localized the attachment molecules to the terminal part of the flagellum. Inhibition of attachment by coating the culture tubes with hydrophilic agarose and constant agitation decreased the rate of epimastigote to trypomastigote transformation, whereas wax coating enhanced metacyclogenesis.

Animals↗
PubMed2002

Making worm guts: the gene regulatory network of the Caenorhabditis elegans endoderm.

The nematode Caenorhabditis elegans is a triploblastic ecdysozoan, which, although it contains too few cells during embryogenesis to create discernible germ "layers," deploys similar programs for germ layer differentiation used in animals with many more cells. The endoderm arises from a single progenitor, the E cell, and is selected from among three possible fates by a three-state combinatorial regulatory system involving intersecting cell-intrinsic and intercellular signals. The core gene regulatory cascade that drives endoderm development, extending from early maternal regulators to terminal differentiation genes, is characterized by activation of successive tiers of transcription factors, including a sequential cascade of redundant GATA transcription factors. Each tier is punctuated by a cell division, raising the possibility that intercession of one cell cycle round, or DNA replication, is required for activation of the next tier. The existence of each tier in the regulatory hierarchy is justified by the assignment of a unique task and each invariably performs at least two functions: to activate the regulators in the next tier and to perform one other activity distinct from that of the next tier. While the regulatory inputs that initiate endoderm development are highly divergent, they mobilize a gene regulatory network for endoderm development that appears to be common to all triploblastic metazoans. Genome-wide functional genomic approaches, including identification of >800 transcripts that exhibit the same regulatory patterns as a number of endoderm-specific genes, are contributing to elucidation of the complete endoderm gene regulatory network in C. elegans. Dissection of the architecture of the C. elegans endoderm network may provide insights into the evolutionary plasticity and origins of this germ layer.

Animals↗
PubMed1989

Anatomical study of the connections of the primary auditory area in the rat.

The aim of the present study was to identify in the rat the overall input-output pattern of connections of the primary auditory field, with special attention to the topographical organization of the geniculocortical auditory projection. By using cytoarchitectural criteria, three temporal cortical fields were distinguished in the rat: Te1, Te2, and Te3. The primary auditory field Te1 is characterized by a relatively specific differentiation of its layers when compared with other temporal fields. The afferent and efferent connections of Te1 were identified by using the retrograde and anterograde transport of wheat germ agglutinin conjugated with horseradish peroxidase (WGA-HRP). The results indicate that Te1 is connected by a dense and reciprocal system of fibers with the auditory thalamus. Based on the nomenclature of Morest ('64) in the cat, five cytoarchitectural subdivisions of the medial geniculate complex (MG) were identified in the rat: ventral (MGv), dorsal (MGd), medial (MGm), suprageniculate (Sg), and peripeduncular (PPA). The major rostrocaudal extent of the MGv is connected to Te1. The surrounding cortical fields Te2 and Te3 do not receive a projection from the MGv, except from its most caudal pole. The MGv projection is topographically organized. When the deposit area of the tracer is shifted from dorsal to ventral upon Te1, the corresponding labeled zone within the MGv moves from rostral to caudal, whereas a cortical displacement of the deposit area of the tracer from rostrodorsal to caudoventral leads to a medial to lateral shift of the labeled zone in the MGv. In addition, more dorsal parts of the MGv project on more dorsal sectors of Te1. Te1 receives a sparser, topographically organized projection from the deep dorsal subdivision of the MGd. The MGm and the lateral part of the posterior group of thalamic nuclei (Pol) also distribute fibers to the primary auditory field. Te1 is reciprocally connected by a system of callosal fibers with the contralateral homotypic cortex. Finally, Te1 sends fibers to the dorsal and, to a lesser extent, external cortices of the inferior colliculus, caudomedial caudate-putamen complex, and caudoventral thalamic reticular nucleus.

Afferent Pathways↗
PubMed2000

Invasion of Candida albicans correlates with expression of secreted aspartic proteinases during experimental infection of human epidermis.

Secreted aspartic proteinases (Saps) encoded by 10 genes of Candida albicans are important virulence factors for different types of candidiasis. Distinct SAP genes have previously been shown to contribute to tissue damage in a model of oral candidiasis. In this study a progressive SAP expression in the order SAP1 and SAP2 > SAP8 > SAP6 > SAP3 was observed in an in vitro model of cutaneous candidiasis based on reconstituted human epidermis. Transcripts of SAP1 and SAP2 were detected during initial invasion of the stratum corneum by C. albicans. Deeper, extensive penetration of the corneal layer was accompanied by additional SAP8 mRNA. SAP6 expression occurred concomitantly with germ tube formation and extensive hyphal growth in the strata granulosum, spinosum, and basale. Ultrastructural studies using specific polyclonal antibodies directed against the gene products of SAP1-3 and SAP4-6 revealed predominant expression of Sap1-3. The protective effect of the aspartic proteinase inhibitor pepstatin A during infection of the epidermis and an attenuated virulence phenotype of SAP-deficient mutants suggest that the observed SAP expression correlates with tissue damage in the skin.

Aspartic Acid Endopeptidases↗
PubMed1999

Maturation ameloblasts of the porcine tooth germ do not express amelogenin.

Amelogenins are the most abundant constituent in the enamel matrix of developing teeth. Recent investigations of rodent incisors and molar tooth germs revealed that amelogenins are expressed not only in secretory ameloblasts but also in maturation ameloblasts, although in relatively low levels. In this study, we investigated expression of amelogenin in the maturation stage of porcine tooth germs by in situ hybridization and immunocytochemistry. Amelogenin mRNA was intensely expressed in ameloblasts from the differentiation to the transition stages, but was not detected in maturation stage ameloblasts. C-terminal specific anti-amelogenin antiserum, which only reacts with nascent amelogenin molecules, stained ameloblasts from the differentiation to the transition stages. This antiserum also stained the surface layer of immature enamel at the same stages. At the maturation stage, no immunoreactivity was found within the ameloblasts or the immature enamel. These results indicate that, in porcine tooth germs, maturation ameloblasts do not express amelogenins, suggesting that newly secreted enamel matrix proteins from the maturation ameloblast are not essential to enamel maturation occurring at the maturation stage.

Ameloblasts↗
PubMed2000

Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers.

BACKGROUND: Embryonic stem (ES) cells are lines of cells that are isolated from blastocysts. The murine ES cells were demonstrated to be true pluripotent cells as they differentiate into all embryonic lineages. Yet, in vitro differentiation of rhesus ES cells was somewhat inconsistent and disorganized. The recent isolation of human ES cells calls for exploring their pluripotential nature. MATERIALS AND METHODS: Human ES cells were grown in suspension to induce their differentiation into embryoid bodies (EBs). The differentiation status of the human ES cells and EBs was analyzed by following the expression pattern of several lineage-specific molecular markers using reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization. RESULTS: Here we report the induction in vitro of cystic embryoid bodies from human ES cells. Our findings demonstrate induction of expression of cell-specific genes during differentiation of the human ES cells into EBs. In the human EBs, we could show a characteristic regional expression of embryonic markers specific to different cellular lineages, namely, zeta-globin (mesoderm), neurofilament 68Kd (ectoderm), and alpha-fetoprotein (endoderm). Moreover, we present a synchronously pulsing embryoid body that expresses the myocardium marker alpha-cardiac actin. In addition, dissociating the embryoid bodies and plating the cells as monolayers results in multiple morphologies, among them cells with neuronal appearance that express neurofilament 68Kd chain. CONCLUSION: Human ES cells can reproducibly differentiate in vitro into EBs comprising the three embryonic germ layers. The ability to induce formation of human embryoid bodies that contain cells of neuronal, hematopoietic and cardiac origins will be useful in studying early human embryonic development as well as in transplantation medicine.

Animals↗
PubMed2005

Stem cell characteristics of amniotic epithelial cells.

Amniotic epithelial cells develop from the epiblast by 8 days after fertilization and before gastrulation, opening the possibility that they might maintain the plasticity of pregastrulation embryo cells. Here we show that amniotic epithelial cells isolated from human term placenta express surface markers normally present on embryonic stem and germ cells. In addition, amniotic epithelial cells express the pluripotent stem cell-specific transcription factors octamer-binding protein 4 (Oct-4) and nanog. Under certain culture conditions, amniotic epithelial cells form spheroid structures that retain stem cell characteristics. Amniotic epithelial cells do not require other cell-derived feeder layers to maintain Oct-4 expression, do not express telomerase, and are nontumorigenic upon transplantation. Based on immunohistochemical and genetic analysis, amniotic epithelial cells have the potential to differentiate to all three germ layers--endoderm (liver, pancreas), mesoderm (cardiomyocyte), and ectoderm (neural cells) in vitro. Amnion derived from term placenta after live birth may be a useful and noncontroversial source of stem cells for cell transplantation and regenerative medicine.

Amnion↗
PubMed1995

Isolation of a primate embryonic stem cell line.

Embryonic stem cells have the ability to remain undifferentiated and proliferate indefinitely in vitro while maintaining the potential to differentiate into derivatives of all three embryonic germ layers. Here we report the derivation of a cloned cell line (R278.5) from a rhesus monkey blastocyst that remains undifferentiated in continuous passage for > 1 year, maintains a normal XY karyotype, and expresses the cell surface markers (alkaline phosphatase, stage-specific embryonic antigen 3, stage-specific embryonic antigen 4, TRA-1-60, and TRA-1-81) that are characteristic of human embryonal carcinoma cells. R278.5 cells remain undifferentiated when grown on mouse embryonic fibroblast feeder layers but differentiate or die in the absence of fibroblasts, despite the presence of recombinant human leukemia inhibitory factor. R278.5 cells allowed to differentiate in vitro secrete bioactive chorionic gonadotropin into the medium, express chorionic gonadotropin alpha- and beta-subunit mRNAs, and express alpha-fetoprotein mRNA, indicating trophoblast and endoderm differentiation. When injected into severe combined immunodeficient mice, R278.5 cells consistently differentiate into derivatives of all three embryonic germ layers. These results define R278.5 cells as an embryonic stem cell line, to our knowledge, the first to be derived from any primate species.

Animals↗
PubMed1993

The Drosophila melanogaster developmental gene g1 encodes a variant zinc-finger-motif protein.

In Drosophila melanogaster, the mechanisms involved in the pattern formation of complex internal organs are still largely unknown. However, the identity of the molecular determinants that control the development of these specific tissues is emerging from the combined use of genetic and molecular approaches. We have cloned a gene that is expressed in the mesoderm, one of the fundamental embryonic germ layers which gives rise to internal structures, such as the musculature. Here, we describe the molecular characterization of this gene, designated as g1. The nucleotide (nt) sequence of its cDNA shows an open reading frame of 852 nt, which encodes a 32-kDa protein with two putative zinc fingers, and a serine/glutamine/proline-rich region. These features indicate a functional role for g1, which remains to be elucidated, in regulating gene expression during mesoderm formation.

Amino Acid Sequence↗
PubMed1989

Expression of laminin and of a laminin-related antigen during early development of Drosophila melanogaster.

This paper reports the characterization of two immunologically related proteins that may be involved in cell adhesion during Drosophila development. These proteins, laminin chain A and a 240K component, share the epitope recognized by monoclonal antibody RD3 (Mab RD3). The two antigens show different developmental expression profiles. Laminin is detected only from 6 to 8 h of development onwards; its concentration increases during embryogenesis to reach steady-state value in larvae, pupae and adult flies. By contrast, the 240K antigen, not found in oocytes, is present before blastoderm stages; its concentration increases during gastrulation, decreases at the end of organogenesis and the antigen is no longer detected in third instar larvae. Light and electron microscope immunolocalization in imaginal discs indicates that laminin is distributed apically in the lumen and basally in the basal membrane that surrounds the nonevaginated disc. During morphogenesis laminin is detected at the basal side of the evaginating part of the disc epithelium. Immunolocalization on paraffin sections of early embryos suggests that the 240K antigen is related to (1) cell formation and polarization in association with cytoskeleton components, (2) establishment of cell-extracellular substratum interactions during the blastoderm cell sheet organization and (3) basement membrane deposition during embryonic germ cell layer segregation. This 240K protein is poorly or not glycosylated, is resistant to chondroitinase ABC and collagenase and appears therefore as a new extracellular component that might be specifically involved in early processes of morphogenesis.

Animals↗
PubMed2002

Fetal brain progenitor cells transdifferentiate to fates outside the nervous system.

Central nervous system stem cells give rise to neurons and glia when exposed to specific trophic factors. In our studies with rat fetal brain-derived stem cells (RSCs), we showed that they could be induced to express the developmentally regulated transcription factors and cell markers characteristic of cells derived from another germ layer, e.g. pituitary cells. Therefore, rat fetal brain-derived stem cells do not seem to be restricted to a defined developmental fate. They may retain pluripotentiality and can be redirected to develop into other cell types not found in the brain provided the correct set of stimuli is present. This multipotent developmental behavior also suggests that instructive signals are operative.

Animals↗
PubMed1988

A mouse pluripotent embryonal stem cell line stage-specifically regulates expression of homeo-box containing DNA sequences during differentiation in vitro.

Mouse embryonal stem (ES) cells have been shown to provide a new model system suitable for the analysis of different aspects of murine development. This report gives evidence that ES cell lines are also most useful for the study of developmentally regulated gene expression in vitro. Homeo-box containing genes which are suggested to play a key role in the regulation of differentiation steps occurring during embryogenesis are stage-specifically transcribed in differentiating murine ES cells: (i) A mouse embryonal stem cell line (ES-12957) was isolated and characterized with respect to its differentiation potential. When injected subcutaneously into syngeneic mice, ES-12957 cells formed fully differentiated teratomas representing derivatives of all three germ layers. When allowed to grow in suspension cultures in vitro, the cells followed a reproducible developmental pathway forming complex organized 'embryoid bodies' which resembled mouse early postimplantation embryos. (ii) A mouse DNA sequence with homeo-box homology (MH-121) was isolated and structurally analyzed. Transcription of a 1.7 kb RNA species from this DNA sequence was demonstrated in ES-12957 cells which were differentiated in vitro. A second, previously described homeo-box gene (Mo-10) was also shown to be expressed in ES-12957 cells in a stage-specific manner. A 4-kb transcript could be identified exclusively in RNA of cells which were allowed to differentiate for 9 days. These findings support the suggestion that the homeo-box genes of mammals, like those of Drosophila, may have important functions during embryonic development.

Animals↗
PubMed2027

Teratoma Formation and Genomic Profiling Using Multi-Omics Approaches.

Teratoma formation is the gold standard assay for evaluating the developmental pluripotency of human and mouse embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Following subcutaneous injection into immunodeficient mice, pluripotent stem cells spontaneously differentiate into derivatives representing all three embryonic germ layers-ectoderm, mesoderm, and endoderm. Beyond serving as a functional assay for pluripotency, teratomas provide a unique three-dimensional model system for studying early human development and lineage specification in vivo. This chapter describes comprehensive protocols for teratoma formation in immunodeficient mice, tissue processing for multiple downstream genomic applications, and multi-omics profiling approaches. We detail methods for embryonic stem cell culture, teratoma generation via subcutaneous injection, tissue dissection and processing for chromatin immunoprecipitation followed by sequencing (ChIP-Seq), RNA sequencing (RNA-Seq), single-cell multiome profiling combining chromatin accessibility (ATAC-Seq) and gene expression (scRNA-Seq), and histological analysis using hematoxylin and eosin (H&E) staining. Additionally, we provide bioinformatics workflows for analyzing the resulting genomic datasets to characterize the epigenetic and transcriptional landscapes of teratoma-derived tissues. These methods enable comprehensive molecular characterization of developmental processes and provide valuable resources for stem cell biologists studying pluripotency, differentiation, and early embryonic development.

Teratoma↗
PubMed1999

Induction of apoptosis in the germline and follicle layer of Drosophila egg chambers.

The reaper and head involution defective genes can induce apoptotic death in several Drosophila cell types, including portions of the embryo and eye. By a combination of FLP recombinase and the yeast Gal4/UAS transcription activation system, we expressed both cell death genes in discrete clones in the adult ovarian follicle cell layer. The expression of either reaper or head involution defective induced follicle cell apoptosis during all oogenic stages. Unexpectedly, the disruption of the follicle layer led to the induced degeneration of the nurse cells in an apoptotic manner, demonstrating a germline-somatic interaction required for germ cell viability. The germline apoptosis initiates at a specific time in oogenesis, coinciding with the beginning of vitellogenesis. This observation is intriguing given previous suggestions of a process to eliminate defective egg chambers at these same oogenic stages. The induce germline degeneration initiates with the transient formation of a network of filamentous actin around the nurse cell nucleus, in close association with a product of the adducin-related hu-li tai shao gene. This was immediately followed by nuclear condensation and DNA fragmentation, both characteristics diagnostic of apoptosis. Occurring concomitantly with the nuclear phenotypes were the disorganization of ring canals, and the degradation of Armadillo protein (a beta-catenin homolog) and filamentous actin. Germ cells degenerating as a normal consequence of oogenesis displayed a similar set of phenotypes, suggesting that a common apoptotic mechanism may underlie these different germline death phenomena.

Actins↗
PubMed2000

The zebrafish forkhead transcription factor FoxH1/Fast1 is a modulator of nodal signaling required for organizer formation.

BACKGROUND: Signaling molecules related to the Nodal protein play essential roles in the formation and patterning of the gastrula organizer and the germ layers during vertebrate development. The forkhead transcription factor FoxH1 (also known as Fast1) is a component of the Nodal signaling pathway. Although different roles have been suggested for FoxH1, its specific function during development is still unclear. RESULTS: We report that the zebrafish locus schmalspur (sur) encodes a member of the FoxH1 family. Maternal sur transcripts were localized to the animal pole during oogenesis. Further expression was detected in a dorsoventral gradient at the onset of gastrulation and in specific domains in the organizer, notochord and lateral plate mesoderm. Embryos lacking zygotic sur function had variable deficiencies of prechordal plate and ventral neuroectoderm. In the absence of both maternal and zygotic sur function, embryos failed to form a morphologically distinct gastrula organizer and, later, developed severe defects in all axial structures. In these embryos, expression of nodal genes was initiated but not maintained. Unlike embryos lacking Nodal signaling, sur mutants formed endoderm and paraxial mesoderm. CONCLUSIONS: FoxH1 is involved in regulatory feedback loops that control the duration and intensity of Nodal signals in early patterning. In zebrafish, FoxH1 is not essential to induce Nodal-dependent cell fates, but its function is central in modulating and enhancing morphogenetic Nodal signals.

Amino Acid Sequence↗