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Results for “germ layer specification”

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PubMed1986

Epidermolysis bullosa acquisita antigen, a major cutaneous basement membrane component, is synthesized by human dermal fibroblasts and other cutaneous tissues.

The epidermolysis bullosa acquisita (EBA) antigen is identified as 2 chains: a 290,000-dalton protein and a less prominent 145,000-dalton protein. The 290,000-dalton chain is synthesized by human keratinocytes in culture. In this study, we show that the 290,000-dalton chain is synthesized by human skin fibroblasts and cutaneous human tumors. In contrast, HT1080 cells, a human sarcoma cell line known to produce matrix molecules (such as laminin and type IV collagen), does not synthesize the EBA antigen. Further, the EBA antigen is absent from serum and blood components, placenta, amnion, lung, and the EHS tumor, a murine sarcoma that produces large amounts of laminin, type IV collagen, nidogen, entactin, and basement membrane proteoglycan but is present in cutaneous tumors of adnexal and epithelial origin. These data suggest that while the EBA antigen is synthesized by both human skin keratinocytes and fibroblasts and is therefore not specific for a primordial germ layer, it does appear to be specific for tissue containing a stratified squamous epithelium.

Antigens↗

PubMed2004

Adult reserve stem cells and their potential for tissue engineering.

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher orders. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50-70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Animals↗

PubMed1991

Immunohistochemical localization of TGF beta 1, TGF beta 2, and TGF beta 3 in the mouse embryo: expression patterns suggest multiple roles during embryonic development.

Isoform-specific antibodies to TGF beta 1, TGF beta 2, and TGF beta 3 proteins were generated and have been used to examine the expression of these factors in the developing mouse embryo from 12.5-18.5 d post coitum (d.p.c.). These studies demonstrate the initial characterization of both TGF beta 2 and beta 3 in mammalian embryogenesis and are compared with TGF beta 1. Expression of one or all three TGF beta proteins was observed in many tissues, e.g., cartilage, bone, teeth, muscle, heart, blood vessels, lung, kidney, gut, liver, eye, ear, skin, and nervous tissue. Furthermore, all three TGF beta proteins demonstrated discrete cell-specific patterns of expression at various stages of development and the wide variety of tissues expressing TGF beta proteins represent all three primary embryonic germ layers. For example, specific localization of TGF beta 1 was observed in the lens fibers of the eye (ectoderm), TGF beta 2 in the cortex of the adrenal gland (mesoderm), and TGF beta 3 in the cochlear epithelium of the inner ear (endoderm). Compared to the expression of TGF beta mRNA transcripts in a given embryonic tissue, TGF beta proteins were frequently colocalized within the same cell type as the mRNA, but in some cases were observed to localize to different cells than the mRNA, thereby indicating that a complex pattern of transcription, translation, and secretion for TGF beta s 1-3 exists in the mouse embryo. This also indicates that TGF beta 1, beta 2, and beta 3 act through both paracrine and autocrine mechanisms during mammalian embryogenesis.

Animals↗

PubMed1999

Delta-mediated specification of midline cell fates in zebrafish embryos.

BACKGROUND: Fate mapping studies have shown that progenitor cells of three vertebrate embryonic midline structures - the floorplate in the ventral neural tube, the notochord and the dorsal endoderm - occupy a common region prior to gastrulation. This common region of origin raises the possibility that interactions between midline progenitor cells are important for their specification prior to germ layer formation. RESULTS: One of four known zebrafish homologues of the Drosophila melanogaster cell-cell signaling gene Delta, deltaA (dlA), is expressed in the developing midline, where progenitor cells of the ectodermal floorplate, mesodermal notochord and dorsal endoderm lie close together before they occupy different germ layers. We used a reverse genetic strategy to isolate a missense mutation of dlA, dlAdx2, which coordinately disrupts the development of floorplate, notochord and dorsal endoderm. The dlAdx2 mutant embryos had reduced numbers of floorplate and hypochord cells; these cells lie above and beneath the notochord, respectively. In addition, mutant embryos had excess notochord cells. Expression of a dominant-negative form of Delta protein driven by mRNA microinjection produced a similar effect. In contrast, overexpression of dlA had the opposite effect: fewer trunk notochord cells and excess floorplate and hypochord cells. CONCLUSION: Our results indicate that Delta signaling is important for the specification of midline cells. The results are most consistent with the hypothesis that developmentally equivalent midline progenitor cells require Delta-mediated signaling prior to germ layer formation in order to be specified as floorplate, notochord or hypochord.

Animals↗

PubMed2006

MicroRNA expression during chick embryo development.

MicroRNAs (miRNAs) are small, abundant, noncoding RNAs that modulate protein abundance by interfering with target mRNA translation or stability. miRNAs are detected in organisms from all domains and may regulate 30% of transcripts in vertebrates. Understanding miRNA function requires a detailed determination of expression, yet this has not been reported in an amniote species. High-throughput whole mount in situ hybridization was performed on chicken embryos to map expression of 135 miRNA genes including five miRNAs that had not been previously reported in chicken. Eighty-four miRNAs were detected before day 5 of embryogenesis, and 75 miRNAs showed differential expression. Whereas few miRNAs were expressed during formation of the primary germ layers, the number of miRNAs detected increased rapidly during organogenesis. Patterns highlighted cell-type, organ or structure-specific expression, localization within germ layers and their derivatives, and expression in multiple cell and tissue types and within sub-regions of structures and tissues. A novel group of miRNAs was highly expressed in most tissues but much reduced in one or a few organs, including the heart. This study presents the first comprehensive overview of miRNA expression in an amniote organism and provides an important foundation for investigations of miRNA gene regulation and function.

Animals↗

PubMed1996

A fate map of the vegetal plate of the sea urchin (Lytechinus variegatus) mesenchyme blastula.

Previous lineage tracing experiments have shown that the vegetal blastomers of cleavage stage embryos give rise to all the mesoderm and endoderm of the sea urchin larva. In these studies, vegetal blastomers were labeled no later than the sixth cleavage division (60-64 cell stage). In an earlier study we showed that single cells in the vegetal plate of the blastula stage Lytechinus variegatus embryo could be labeled in situ with the fluorescent, lipophilic dye, DiI(C18), and that cells labeled in the central region of the vegetal plate of the mesenchyme blastula primarily gave rise to homogeneous clones consisting of a single secondary mesenchyme cell (SMC) type (Ruffins and Ettensohn (1993) Dev. Biol. 160, 285-288). Our clonal labeling showed that a detailed fate map could be generated using the DiI(C18) labeling technique. Such a fate map could provide information about the spatial relationships between the precursors of specific mesodermal and endodermal cell types and information concerning the movements of these cells during gastrulation and later embryogenesis. We have used this method to construct the first detailed fate map of the vegetal plate of the sea urchin embryo. Ours is a latitudinal map; mapping from the plate center, where the mesodermal precursors reside, through the region which contains the endodermal precursors and across the ectodermal boundary. We found that the precursors of certain SMC types are segregated in the mesenchyme blastula stage vegetal plate and that prospective germ layers reside within specific boundaries. To determine whether the vegetal plate is radially symmetrical with respect to mesodermal cell fates, single blastomeres of four cell stage embryos were injected with lysyl-rhodamine dextran (LRD). The resulting ectodermal labeling patterns were classified and correlated with the SMC types labeled. This analysis indicates that the dorsal and ventral blastomers do not contribute equally to SMC derivatives in L. variegatus.

Animals↗

PubMed2004

Adult-derived liver stem cells acquire a cardiomyocyte structural and functional phenotype ex vivo.

We examined the differentiation potential of an adult liver stem cell line (WB F344) in a cardiac microenvironment, ex vivo. WB F344 cells were established from a single cloned nonparenchymal epithelial cell isolated from a normal male adult rat liver. Genetically modified, WB F344 cells that express beta-galactosidase and green fluorescent protein or only beta-galactosidase were co-cultured with dissociated rat or mouse neonatal cardiac cells. After 4 to 14 days, WB F344-derived cardiomyocytes expressed cardiac-specific proteins and exhibited myofibrils, sarcomeres, and a nascent sarcoplasmic reticulum. Further, rhythmically beating WB F344-derived cardiomyocytes displayed calcium transients. Fluorescent recovery after photobleaching demonstrated that WB F344-derived cardiomyocytes were coupled with adjacent neonatal cardiomyocytes and other WB F344-derived cardiomyocytes. Fluorescence in situ hybridization experiments suggested that fusion between WB F344 cells and neonatal mouse cardiomyocytes did not take place. Collectively, these results support the conclusion that these adult-derived liver stem cells respond to signals generated in a cardiac microenvironment ex vivo acquiring a cardiomyocyte phenotype and function. The identification ex vivo of microenvironmental signals that appear to cross germ layer and species specificities should prove valuable in understanding the molecular basis of adult stem cell differentiation and phenotypic plasticity.

Animals↗

PubMed2003

Maternal factors in zebrafish development.

All processes that occur before the activation of the zygotic genome at the midblastula transition are driven by maternal products, which are produced during oogenesis and stored in the mature oocyte. Upon egg activation and fertilization, these maternal factors initiate developmental cascades that carry out the embryonic developmental program. Even after the initiation of zygotic gene expression, perduring maternal products continue performing essential functions, either together with other maternal factors or through interactions with newly expressed zygotic products. Advances in zebrafish research have placed this organism in a unique position to contribute to a detailed understanding of the role of maternal factors in early vertebrate development. This review summarizes our knowledge on the processes involved in the production and redistribution of maternal factors during zebrafish oogenesis and early development, as well as our understanding of the function of these factors in axis formation, germ layer and germ cell specification, and other early embryonic processes.

Animals↗

PubMed1989

SPARC: a Ca2+-binding extracellular protein associated with endothelial cell injury and proliferation.

SPARC (Secreted Protein that is Acidic and Rich in Cysteine) is a Ca2+-binding, stress-related protein released in vitro by both malignant and normal cells derived from all primordial germ layers. It is specifically elevated in endothelial cells as a result of "culture shock" (characterized by high levels of proliferation and migration) and exhibits density-dependent secretion. Exposure of bovine aortic endothelial cells to endotoxin results in a 70-100% increase in secreted protein, with a three-fold increase in SPARC. Immunofluorescence histochemistry on mouse tissues revealed (a) a preferential association of SPARC with highly proliferative cells (e.g., gut epithelia, mammary gland, and epidermis), (b) a cell surface or stromal location in thymus, lung, and salivary gland, (c) staining of epididymidal epithelium and testicular cells, and (d) an association with somites of 14 d mouse embryos. We envision SPARC as an extracellular modulator of Ca2+ and other cation-sensitive proteins/proteinases, that facilitates cellular proliferation in response to injury and to developmental signals.

Animals↗

PubMed1996

heartless encodes a fibroblast growth factor receptor (DFR1/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophila embryo.

After invagination of the mesodermal primordium in the gastrulating Drosophila embryo, the internalized cells migrate in a dorsolateral direction along the overlying ectoderm. This movement generates a stereotyped arrangement of mesodermal cells that is essential for their correct patterning by later position-specific inductive signals. We now report that proper mesodermal cell migration is dependent on the function of a fibroblast growth factor (FGF) receptor encoded by heartless (htl). In htl mutant embryos, the mesoderm forms normally but fails to undergo its usual dorsolateral migration. As a result, cardiac, visceral, and dorsal somatic muscle fates are not induced by Decapentaplegic (Dpp), a transforming growth factor beta family member that is derived from the dorsal ectoderm. Visceral mesoderm can nevertheless be induced by Dpp in the absence of htl function. Ras1 is an important downstream effector of Htl signaling because an activated form of Ras1 partially rescues the htl mutant phenotype. The evolutionary conservation of htl function is suggested by the strikingly similar mesodermal migration and patterning phenotypes associated with FGF receptor mutations in species as diverse as nematode and mouse. These studies establish that Htl signaling provides a vital connection between initial formation of the embryonic mesoderm in Drosophila and subsequent cell-fate specification within this germ layer.

Alleles↗

PubMed2004

Development of definitive endoderm from embryonic stem cells in culture.

The cellular and molecular events regulating the induction and tissue-specific differentiation of endoderm are central to our understanding of the development and function of many organ systems. To define and characterize key components in this process, we have investigated the potential of embryonic stem (ES) cells to generate endoderm following their differentiation to embryoid bodies (EBs) in culture. We found that endoderm can be induced in EBs, either by limited exposure to serum or by culturing in the presence of activin A (activin) under serum-free conditions. By using an ES cell line with the green fluorescent protein (GFP) cDNA targeted to the brachyury locus, we demonstrate that endoderm develops from a brachyury(+) population that also displays mesoderm potential. Transplantation of cells generated from activin-induced brachyury(+) cells to the kidney capsule of recipient mice resulted in the development of endoderm-derived structures. These findings demonstrate that ES cells can generate endoderm in culture and, as such, establish this differentiation system as a unique murine model for studying the development and specification of this germ layer.

Activins↗

PubMed1999

Pax genes and their role in organogenesis.

Pax genes have been cloned on the basis of their homology to the Drosophila segmentation gene paired. They share a common domain, the paired domain, that is sufficient to mediate sequence-specific DNA binding. Thus far, nine members have been characterized, which exhibit highly restricted temporal and spatial expression patterns. The analysis of mouse mutants has revealed their crucial role in the formation of a variety of tissues. In particular, they are involved in the regulation of early steps in organ development. They act to define the regional specification of distinct germ layers.

Animals↗

PubMed2004

Ectodermal dysplasias.

Ectodermal dysplasias are a large group of heritable conditions characterized by congenital defects of one or more ectodermal structures and their appendages: hair (hypotrichosis, partial, or total alopecia), nails (dystrophic, hypertrophic, abnormally keratinized), teeth (enamel defect or absent), and sweat glands (hypoplastic or aplastic). The ectodermal dysplasias, as a rule, are not pure "one-layer diseases." Mesodermal and, rarely, endodermal dysplasias coexist. Embryogenesis exhibits distinct tissue organizational fields and specific interactions among the germ layers that may lead to a wide range of ectodermal dysplasias when genes important for development are mutated or otherwise altered in expression. Of the approximately 200 different ectodermal dysplasias, about 30 have been studied at the molecular level with identification of the causative gene. Freire-Maia and Pinheiro used the clinical aspects for their classification, and Priolo integrated molecular genetic and clinical aspects for her scheme. Those two more historical classification schemes have the difficulty that, when applied strictly, several additional groups of diseases should be integrated within the term "ectodermal dysplasias," e.g. keratodermas with skin or hair alterations or the ichthyoses with associated features. Such consequent classification would lead to an endless list of diseases and would be useless for the practical work. Recent evidence implicates a genetic defect in different pathways orchestrating ectodermal organogenesis. Modern molecular genetics will increasingly elucidate the basic defects of the different syndromes and yield more insight into the regulatory mechanisms of embryology. In this way, a reclassification of ectodermal dysplasias will be possible according to the function of their involved mutated genes. Lamartine recently proposed a helpful classification according to the functions of the genes discovered in different types of ectodermal dysplasias. Accordingly, the present overview categorizes the various ectodermal dysplasias into four major functional subgroups: cell-cell communication and signaling, adhesion, transcription regulation, and development.

Cell Adhesion↗

PubMed2006

Derivation of distal lung epithelial progenitors from murine embryonic stem cells using a novel three-step differentiation protocol.

Embryonic stem cells (ESCs) are a potential source for the cell-based therapy of a wide variety of lung diseases for which the only current treatment is transplantation. However, distal lung epithelium, like many other endodermally derived somatic cell lineages, is proving difficult to obtain from both murine and human ESCs. We have previously obtained alveolar epithelium from ESCs, although final cell yield remained extremely low. Here, we present an optimized three-step protocol for the derivation of distal lung epithelial cells from murine ESCs. This protocol incorporates (a) treatment of early differentiating embryoid bodies with activin A to enhance the specification of the endodermal germ layer, followed by (b) adherent culture in serum-free medium and (c) the final application of a commercial, lung-specific medium. As well as enhancing the specification of distal lung epithelium, this protocol was found to yield cells with a phenotype most closely resembling that of lung-committed progenitor cells present in the foregut endoderm and the early lung buds during embryonic development. This is in contrast to our previous differentiation method, which drives differentiation through to mature type II alveolar epithelial cells. The derivation of a committed lung progenitor cell type from ESCs is particularly significant for regenerative medicine because the therapeutic implantation of progenitor cells has several clear advantages over the transplantation of mature, terminally differentiated somatic cells.

Activins↗

PubMed1985

Normal fates and states of specification of different regions in the axolotl gastrula.

A fate map was constructed for four regions of the early gastrula of Ambystoma mexicanum using orthotopic grafts from donors labelled with FLDx (fluoresceinated-lysinated-dextran). The region around the animal pole gave rise to epidermis only and did not include prospective neural plate. The dorsal marginal zone contributed to cephalic endoderm and to the whole length of the axial mesoderm (notochord and somites), the lateral marginal zone to lateroventral and somitic mesoderm, and the ventral marginal zone to lateroventral mesoderm. It was found that the dorsal marginal zone contributed relatively more to the anterior regions of the mesodermal mantle and the ventral marginal zone more to its posterior parts. The same regions of the gastrula and also vegetal yolky tissue were cultured as explants and labelled with tritiated mannose. Their glycoprotein synthesis pattern was compared to those of the neurula tissues to which they contribute in vivo. Animal pole explants synthesized large amounts of the epidermis-specific marker epimucin. Dorsal marginal zone explants did not synthesize epimucin but did make amounts of S2 and S6 indicative of mesoderm, as well as the notochord-specific markers S2.2 and S3.2. Lateral marginal zone explants showed the same pattern as the dorsal marginal zone including the two notochord-specific markers, although they do not contribute to notochord in vivo. Ventral marginal zone explants were more variable in their behaviour. Yolky tissue from the vegetal hemisphere of the gastrula or the archenteron floor of the neurula synthesized mainly polydisperse material of high molecular weight rather than discrete glycoproteins. The results indicate that at the early gastrula stage states of specification exist which correspond to the three germ layers, ecto-, meso- and endoderm. The ectodermal specification of animal pole explants is quite robust and cannot easily be changed by variation of the culture conditions. However treatment with a concentrated pellet of vegetalizing factor does induce a change to mesodermal specification, which is clearly detectable in the pattern of glycoprotein synthesis. Similar inductive interactions between different regions of the early embryo are thought to occur during normal development.

Ambystoma mexicanum↗

PubMed1992

Establishment and organization of germ layers in the gastrulating mouse embryo.

By following the distribution of wheat germ agglutinin-gold-labelled cells in primitive streak stage embryos, we obtained direct evidence for a continuous recruitment of the embryonic ectoderm cells to the definitive endoderm and to the embryonic and extraembryonic mesoderm during gastrulation. The majority of the definitive endodermal cells ingressed through the anterior end of the primitive streak and were incorporated initially into the midline endoderm at the archenteron, but a small population of endodermal cells may be recruited by direct delamination from the embryonic ectoderm. The pre-existing visceral embryonic endoderm was progressively replaced, but not totally, by the newly recruited population which colonized the embryonic foregut and the notochord. The developmental fate of the recruited endoderm and that of cells in the embryonic ectoderm and the mesoderm of late primitive streak stage embryos indicate that concomitant with the establishment of the germ layers, an orderly allocation of prospective fetal tissues to specific parts of the body occurs simultaneously in all three germ layers.

Animals↗

PubMed1981

Retrovirus-encoded transformation-specific polyproteins: expression coordinated with malignant phenotype in cells from different germ layers.

A transformation-associated polyprotein designated "gag-x" was previously shown to be induced by the feline sarcoma virus (FeSV) after the nonproductive transformation of rat or mink cells. We found that this protein was also expressed in cells derived from the native species (cat) with or without the production of feline leukemia helper virus (FeLV) and that cats could mount a humoral antibody response to the transformation-specific (x) portion of the molecule. Such antisera also reacted with the feline oncornavirus-associated cell membrane antigen (FOCMA) by membrane immunofluorescence. Expression of the gag-x protein was coordinated with malignant phenotype in that both transformed cat fibroblasts and cultured cells from a FeSV-induced melanoma expressed antigenically indistinguishable proteins of the same size. These cells are derived from different embryonic germ layers, suggesting that such transformation-related proteins may function in a pleiotropic manner when introduced by a virus.

Animals↗

PubMed1993

Testicular degeneration and spermatid retention in young male rats.

The incidence of spontaneous testicular atrophy and its morphological changes in relation to stage-specific spermatogenesis were investigated in young Crl:CD/BR male rats at 10-12 wk of age used as controls for toxicity screening during 1983-1990. The incidence of testicular degeneration was 2.5% (5/197) in control rats used for oral toxicity studies and 9.4% (31/327) in rats used for inhalation studies. The epididymal tubules of rats with testicular degeneration had exfoliated germ cells and low sperm density. The high incidence of testicular degeneration observed in the control rats used in inhalation studies may be related to the stress associated with immobilization in the restrainer during nose-only exposure conditions. The severity of testicular degeneration in the inhalation studies was mostly minimal. In these minimally affected testes, mature spermatids (step 19) were retained within normal-appearing germinal epithelium at spermatogenic stages IX-XIV. Also, eosinophilic globular bodies (EGBs) were formed with elongated or mature spermatids throughout all spermatogenic stages, but the general architecture of germinal epithelium was normal in appearance. By electron microscopy, EGBs were sequestered necrotic spermatids, and the germ cell degeneration was associated with cytoplasmic vacuolation of Sertoli cells. In moderate testicular degeneration, markedly decreased maturing spermatids (steps 15-19) and a slight depletion of round spermatids were observed in stages I-VIII. In severe testicular degeneration, seminiferous tubules were lined with 1-2 layers of round spermatids and spermatocytes with giant cell formation. The round spermatids served as a marker to identify spermatogenic stages (I-VIII) of the atrophic tubules. Also, in severe testicular degeneration, tubules in spermatogenic stages X-XIV had no elongated spermatids, and spermatocytes were exfoliated with occasional giant cell formation. Many seminiferous tubules were lined with only 1-2 layers of spermatocytes, and specific germ cell markers were not present.

Administration, Inhalation↗