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PubMed2001

Pluripotency of bovine embryonic cell line derived from precompacting embryos.

We report herein the establishment of three bovine pluripotent embryonic cell lines derived from 8-16-cell precompacting embryos. Two cell lines were cultured for 10 passages and underwent spontaneous differentiation. One cell line (Z2) has been cultured continuously for over 3 years and has remained undifferentiated. These cells express cell surface markers that have been used routinely to characterize embryonic stem (ES) and embryonic germ (EG) cells in other species such as stage-specific embryonic antigens SSEA-1, SSEA-3, and SSEA-4, and c-Kit receptor. In the absence of a feeder layer, these cells differentiated into a variety of cell types and formed embryoid bodies (EBs). When cultured for an extended period of time, EBs differentiated into derivatives of three EG layers - mesoderm, ectoderm, and endoderm - which were characterized by detection of specific cell surface markers. Our results indicate that the Z2 cell line is pluripotent and resembles an ES cell line. To our knowledge, this is the first bovine embryonic cell line that has remained pluripotent in culture for more than 150 passages.

Animals↗
PubMed2006

Mesodermal expression of Tbx1 is necessary and sufficient for pharyngeal arch and cardiac outflow tract development.

The development of the segmented pharyngeal apparatus involves complex interaction of tissues derived from all three germ layers. The role of mesoderm is the least studied, perhaps because of its apparent lack of anatomical boundaries and positionally restricted gene expression. Here, we report that the mesoderm-specific deletion of Tbx1, a T-box transcription factor, caused severe pharyngeal patterning and cardiovascular defects, while mesoderm-specific restoration of Tbx1 expression in a mutant background corrected most of those defects in the mouse. We show that some organs, e.g. the thymus, require Tbx1 expression in the mesoderm and in the epithelia. In addition, these experiments revealed that different pharyngeal arches require Tbx1 in different tissues. Finally, we show that Tbx1 in the mesoderm is required to sustain cell proliferation. Thus, the mesodermal transcription program is not only crucial for cardiovascular development, but is also key in the development and patterning of pharyngeal endoderm.

Animals↗
PubMed1998

[Molecular mechanism of limb muscle patterning].

Pattern formation of the limb muscles involves a network between intrinsic programs of gene expression in myogenic precursor cells and extrinsic signals derived from the surrounding tissue. The cells at the lateral edge of the somite are specified to the precursors of the hypaxial muscles by intercelluler interaction with the dorsal ectoderm and the lateral mesoderm. At the level of the limb bud, these premyogenic cells expressing c-MET and Pax-3 are induced to invade into the limb bud by HGF/SF and unknown chemotactic signal(s) exerted by the limb mesenchyme. It is likely that N-cadherin and some extracellular matrixes are involved in this phenomena. In the limb bud, myogenic cells proliferate under influence of the limb ectoderm and congregate into the premuscle masses. BMPs expressed in limb ectoderm may maintain the proliferative phase of the myogenic cells by inhibition of the myogenic differentiation. Recently, detailed analysis of expression of Hox genes in the chick limb musculature suggests that Hoxa-11 and Hoxa-13 autonomously regulate the pattern formation of the limb muscles. Hoxa-11 is induced in the myogenic cells by an interaction with the limb mesenchyme as soon as they invade into the limb. It is possible that Hoxa-11 may confer a limb-specific property on these myogenic cells. After the formations of muscle masses, Hoxa-11 and Hoxa-13 are expressed in the muscle masses in region-specific manner along the anteroposterior and proximodistal axes under the control of the limb mesenchyme and the polarizing signal(s). They are candidates for intrinsic regulator of myogenic cells that control 1) the splitting processes of the muscle masses and 2) the specification of each muscle.

Animals↗
PubMed1993

Sequences 5' of the homeobox of the Hox-1.4 gene direct tissue-specific expression of lacZ during mouse development.

The murine homeobox-containing gene Hox-1.4 is expressed in restricted patterns during embryogenesis and in male germ cells. To begin identification of the cis-acting elements regulating this expression, transgenic mice were generated carrying a chimeric construct that contained approx. 4 kb of 5' flanking sequence and approx. 1 kb of structural gene, fused in frame to the E. coli lacZ gene. This construct directed expression of the resulting Hox-1.4,beta-galactosidase fusion protein in a pattern that reproduced virtually the complete embryonic and adult sites of expression of the endogenous gene. Embryonic expression of the fusion protein was first detected in mesoderm at day 8.0 of gestation (E 8.0). Between gestational ages E 8.5 to E 12.5, beta-gal expression was observed in the somites, the lateral walls of the posterior myelencephalon, the dorsal region and ventral wall of the spinal cord, spinal ganglia and prevertebrae and their surrounding mesenchyme, between presumptive ribs, as well as in mesenchymal layers in the lung, kidney and portions of the gut. Expression was also noted in the pancreas and in the supporting cells and sheath around subsets of peripheral nerves, sites that had not been detected previously. Adult expression was observed in testes, specifically in meiotic and post-meiotic male germ cells. In contrast, transgenic mice carrying 5' deletions of the construct which leave approx. 1.2 kb or approx. 2.0 kb of Hox-1.4 sequence 5' to the embryonic promoter, did not exhibit beta-gal staining. These deletion experiments defined at least one cis-acting control element necessary for the expression of the Hox-1.4 gene to a 2 kb region located 2 to 4 kb 5' of the embryonic transcription start site.

Animals↗
PubMed1983

Regional distribution of N-acetyl-D-galactosamine residues in the glycocalyx of glomerular podocytes.

Helix pomatia lectin (HPL) bound to colloidal gold was used as a specific cytochemical probe for the localization of terminal nonreducing N-acetyl-D-galactosamine residues in thin sections of rat kidney. In the glomerulus, lectin-binding sites were associated only with the podocyte foot process bases and were not found on the free cell surface of podocytes or on any other glomerular components. Gold-particle label was often arranged in the form of clusters which extended from the foot process base to the lamina rare externa and lamina densa of the basement membrane. In contrast, wheat germ lectin (WGL)-binding sites (beta-[1 leads to 4] linked N-acetyl-D-glucosamine residues and N-acetylneuraminic acid residues) were found in all regions of the podocyte plasma membrane and on the cell surface of all other glomerular cell types. In addition, WGL-binding sites were present in all three layers of the glomerular basement membrane (GBM) as well as in the mesangial matrix. A quantitative evaluation of the pattern of labeling for HPL-binding sites together with the sugar specificity of this lectin suggest that a component of the glycocalyx is being detected rather than a basement membrane component. This was confirmed by the absence of H. pomatia lectin-binding sites in preparations of isolated GBM which retained, however, wheat germ lectin-binding sites. These data show that the glycocalyx of the foot process base is a highly specialized cell surface domain with respect to its carbohydrate composition.

Acetylgalactosamine↗
PubMed1996

Origin of primordial germ cells in the prestreak chick embryo.

The temporal and spatial pattern of segregation of the avian germline from the formation of the area pellucida to the beginning of primitive streak formation (stages VII-XIV, EG&K) was investigated using the culture of whole embryos and central and peripheral embryo fragments on vitelline membranes at stages VII-IX, immunohistological analysis of whole mount embryos and sections with monoclonal antibodies MC-480 against stage-specific embryonic antigen-1 (SSEA-1) and EMA-1, and with the culture of dispersed blastoderms at stages IX-XIV with and without on STO feeder layer. Whole embryos at intrauterine stages developed up to the formation of the primitive streak despite the absence of area pellucida expansion. Primordial germ cells (PGCs) appeared in the cultures of whole embryos and only in central fragments containing a partially formed area pellucida at stages VII-IX. When individual stage IX-XIV embryos were dispersed and cultured without a feeder layer, 25-45 PGCs/embryo were detected only with stage X-XIV, but not with stage IX blastoderms. However, the culture of dispersed cells from the area pellucida of stages IX-XIII on STO feeder layers yielded about 150 PGCs/embryo. The carbohydrate epitopes recognized by anti-SSEA-1 and EMA-1 first appeared at stage X on cells in association with polyingressing cells on the ventral surface of the epiblast and later on the dorsal surface of the hypoblast. The SSEA-1-positive hypoblast cells gave rise to chicken PGCs when cultured on a feeder layer of quail blastodermal cells. From these observations, we propose that the segregation and development of avian germline is a gradual, epigenetic process associated with the translocation of SSEA-1/EMA-1-positive cells from the ventral surface of the area pellucida at stage X to the dorsal side of the hypoblast at stages XI-XIV.

Animals↗
PubMed2000

Regulation of epiblast cell movements by chondroitin sulfate during gastrulation in the chick.

Avian gastrulation is dependent on the ingression of outer layer cells into the interior of the embryo by means of a transient structure referred to as the primitive streak. As the growing streak progresses through the central area pellucida of the blastoderm, selective de-epithelialization of epiblast cells results in the initial migratory cells of the primitive mesoderm and endoderm. Here, we have examined the possibility that extracellular matrix molecules of the epiblast basal lamina influence the selection of streak-specific epiblast cells. By using whole embryo culture, we have found that removal of chondroitin sulfate glycosaminoglycans at gastrulation stages leads to defective streak formation. In situ hybridization with streak-specific markers in these embryos reveals ectopic patterns of gene expression, suggesting that differentiation of primitive streak precursors in the pregastrula epiblast is independent of normal streak morphogenesis. In addition, in vitro assays with chondroitin sulfate containing matrices suggest that specific cells of the epiblast are inhibited from joining the streak during gastrulation. Taken together, these results indicate that the presence of chondroitin sulfate in the epiblast basal lamina facilitates the allocation of cells to the primary germ layers by preventing ectopic axis formation.

Animals↗
PubMed2006

Derivation of human embryonic stem cells from developing and arrested embryos.

Human embryonic stem cells (hESC) hold huge promise in modern regenerative medicine, drug discovery, and as a model for studying early human development. However, usage of embryos and derivation of hESC for research and potential medical application has resulted in polarized ethical debates since the process involves destruction of viable developing human embryos. Here we describe that not only developing embryos (morulae and blastocysts) of both good and poor quality but also arrested embryos could be used for the derivation of hESC. Analysis of arrested embryos demonstrated that these embryos express pluripotency marker genes such OCT4, NANOG, and REX1. Derived hESC lines also expressed specific pluripotency markers (TRA-1-60, TRA-1-81, SSEA4, alkaline phosphatase, OCT4, NANOG, TERT, and REX1) and differentiated under in vitro and in vivo conditions into derivates of all three germ layers. All of the new lines, including lines derived from late arrested embryos, have normal karyotypes. These results demonstrate that arrested embryos are additional valuable resources to surplus and donated developing embryos and should be used to study early human development or derive pluripotent hESC.

Embryo, Mammalian↗
PubMed2003

Multilineage potential of homozygous stem cells derived from metaphase II oocytes.

Human stem cells derived from human fertilized oocytes, fetal primordial germ cells, umbilical cord blood, and adult tissues provide potential cell-based therapies for repair of degenerating or damaged tissues. However, the diversity of major histocompatibility complex (MHC) antigens in the general population and the resultant risk of immune-mediated rejection complicates the allogenic use of established stem cells. We assessed an alternative approach, employing chemical activation of nonfertilized metaphase II oocytes for producing stem cells homozygous for MHC. By using F1 hybrid mice (H-2-B/D), we established stem cell lines homozygous for H-2-B and H-2-D, respectively. The undifferentiated cells retained a normal karyotype, expressed stage-specific embryonic antigen-1 and Oct4, and were positive for alkaline phosphatase and telomerase. Teratomatous growth of these cells displayed the development of a variety of tissue types encompassing all three germ layers. In addition, these cells demonstrated the potential for in vitro differentiation into endoderm, neuronal, and hematopoietic lineages. We also evaluated this homozygous stem cell approach in human tissue. Five unfertilized blastocysts were derived from a total of 25 human oocytes, and cells from one of the five hatched blastocysts proliferated and survived beyond two passages. Our studies demonstrate a plausible "homozygous stem cell" approach for deriving pluripotent stem cells that can overcome the immune-mediated rejection response common in allotransplantation, while decreasing the ethical concerns surrounding human embryonic stem cell research.

Animals↗
PubMed2007

Chordin affects pronephros development in Xenopus embryos by anteriorizing presomitic mesoderm.

Spemann's organizer emits signals that pattern the mesodermal germ layer during Xenopus embryogenesis. In a previous study, we demonstrated that FGFR1 activity within the organizer is required for the production of both the somitic muscle- and pronephros-patterning signals by the organizer and the expression of chordin, an organizer-specific secreted protein (Mitchell and Sheets [2001] Dev. Biol. 237:295-305). Studies from others in both chicken and Xenopus embryos provide compelling evidence that pronephros forms by means of secondary induction signals emitted from anterior somites (Seufert et al. [1999] Dev. Biol. 215:233-242; Mauch et al. [2000] Dev. Biol. 220:62-75). Here we provide several lines of evidence in support of the hypothesis that chordin influences pronephros development by directing the formation of anterior somites. Chordin mRNA was absent in ultraviolet (UV) -irradiated embryos lacking pronepheros (average DAI<2) but was always found in UV-irradiated embryos that retain pronepheros (average DAI>2). Furthermore, ectopic expression of chordin in embryos and in tissue explants leads to the formation of anterior somites and pronephros. In these experiments, pronephros was only observed in association with muscle. Chordin diverted somatic muscle cells to more anterior positions within the somite file in chordin-induced secondary trunks and induced the expression of the anterior myogenic gene myf5. Finally, depletion of chordin mRNA with DEED antisense oligonucleotides substantially reduced somitic muscle and pronephric tubule and duct formation in whole embryos. These data and previous studies on ectoderm and endoderm (Sasai et al. [1995] Nature 377:757) support the idea that chordin functions as an anteriorizing signal in patterning the germ layers during vertebrate embryogenesis. Our data support the hypothesis that chordin directs the formation of anterior somites that in turn are necessary for pronephros development.

Animals↗
PubMed2005

Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes.

Previous reports and the current study have found that germ cell precursor cells appear in embryoid bodies (EBs) formed from mouse embryonic stem cells as identified by positive expression of specific germ cell markers such as Oct-3/4, Mvh, c-kit, Stella, and DAZL. We hypothesized that if exposed to appropriate growth factors, the germ cell precursor cells within the EBs would differentiate into gametes. The source for growth factors used in the present study is conditioned medium collected from testicular cell cultures prepared from the testes of newborn males. Testes at this stage of development contain most growth factors required for the transformation of germ stem cells into differentiated gametes. When EBs were cultured in the conditioned medium, they developed into ovarian structures, which contained putative oocytes. The oocytes were surrounded by one to two layers of flattened cells and did not have a visible zona pellucida. However, oocyte-specific markers such as Fig-alpha and ZP3 were found expressed by the ovarian structures. The production of oocytes using this method is repeatable and reliable and may be applicable to other mammalian species, including the human.

Animals↗
PubMed1984

Characterization of a pluripotent stem cell line derived from a mouse embryo.

A pluripotent, karyotypically normal, male culture line ESC-BLC 1 of embryonal stem cells was established from delayed mouse blastocysts of strain 129/ter Sv. The cell line was isolated after cultivation of inner cell mass cells on X-irradiated feeder layer of mouse embryonal fibroblasts. The pluripotent status of the cell line was confirmed by in vivo and in vitro differentiation. For in vivo differentiation, cells were injected subcutaneously into syngeneic mice. The resulting tumors contained various tissues, derivatives of all three primary germ layers. In vitro cultivated pluripotent stem cells differentiated into endoderm-like, neuronal-like and tubular structures. Determination of alkaline phosphatase in cell line ESC-BLC 1 yielded a high specific activity; G-banding of metaphases revealed a normal, male karyotype.

Alkaline Phosphatase↗
PubMedDate not supplied

Xenopus differentiation: VegT gets specific.

Depletion of the maternal store of the localised mRNA encoding the T-box transcription factor VegT in Xenopus embryos has recently been shown to dramatically block endoderm formation and change the normal position of the mesodermal and ectodermal germ layers.

Animals↗
PubMed1993

The complementary deoxyribonucleic acid sequence, tissue distribution, and cellular localization of the rat granulin precursor.

Granulins (grns; also called epithelins) are cysteine-rich polypeptides with pleiotropic effects on epithelial cell growth in vitro. The grn/epi gene is widely expressed in epithelial cell lines, many of which respond to the gene product, raising the possibility of autocrine or paracrine regulation. In vitro the grn gene is expressed in cell types of diverse lineages, including epithelial cells, lymphoid and myeloid cells, and fibroblasts, but it is not known which cells express the gene in vivo. To understand the physiological role of the grn gene products it is necessary to know the context of grn gene expression in vivo. We have isolated the rat grn precursor complementary DNA and determined, by Northern blot analysis and in situ hybridization, the tissue distribution and cellular localization of grn gene expression. The complementary DNA predicts a 589-amino acid protein of M(r) 63,500 with seven and one-half grn repeats arranged in tandem and shows an overall identity of 75% with human progrn. The grn gene is expressed in a variety of tissues derived from all three embryonic germ layers but is most abundant in the spleen and several tissues of endocrine significance including the adrenal glands, epididymis, placenta, and ovary. Although widely expressed in tissues, gene expression is restricted to specific cell types. For example in the kidney, grn messenger RNA was detected in epithelial cells of the proximal and distal convoluted tubules and Bowman's capsule but not in medullary epithelia. In the spleen, grn messenger RNA expression was localized in lymphocytes, whereas hybridization signals were detected over scattered hepatocytes in the liver. Thus, although the grn gene is widely expressed in tissues and cell lines of many lineages in vitro, its expression in situ is restricted to hematopoietic and some epithelial cells. The restricted cell distribution suggests that the expression of the grn gene is more closely regulated in vivo than in cell cultures. Its localization to epithelial cells in situ supports an autocrine or paracrine role for these factors.

Amino Acid Sequence↗
PubMed1998

Effects of neonatal administration of diethylstilbestrol in male hamsters: disruption of reproductive function in adults after apparently normal pubertal development.

Prenatal and neonatal exposure to natural and synthetic estrogens induces developmental abnormalities in the male and female reproductive systems in several species. In hamsters, a single injection of diethylstilbestrol (DES) on the day of birth induces teratogenic and neoplastic changes throughout the female reproductive tract, apparently via a direct mechanism. The present study investigated the extent and specificity of this phenomenon in the male reproductive system. Male golden hamsters received injections of DES or estradiol-17beta (E2; 100 microg/ animal) on the day of birth and were then killed at 42 (pubertal) and 90 (adults) days of age. Blood was collected for serum testosterone analysis, and the testes and accessory organs were weighed and examined histologically. At the pubertal stage, testicular and accessory organ weights plus serum testosterone levels were similar in untreated animals and in both groups of estrogen-treated animals. Also at the pubertal stage, initiation of spermatogenesis appeared normal in both groups of estrogen-treated animals. In contrast, 100% of the DES-treated animals (n = 22) but none of the E2-treated animals exhibited multiple lesions in the reproductive tract as adults. These DES-induced lesions included cryptorchidism with the testes attached to the abdominal wall, solid testicular tumors, multiple epididymal cysts, and involution of accessory organs. Spermatogenesis was disrupted in the seminiferous tubules, with no developing germ cells, and the interstitial cells were organized as a sheath around the dysfunctional tubules. The epididymis had an involuted epithelial layer with a preponderance of multi-nucleated cells, and seminal vesicle morphology was also abnormal. These DES-specific alterations were not accompanied by any significant change in circulating testosterone levels. We therefore conclude that 1) DES is much more potent that E2 as a neonatal endocrine disrupter in the male hamster, and 2) the DES-specific lesions in the adult male reproductive tract may represent a permanently altered androgen responsiveness in the affected target tissues.

Animals↗
PubMed2005

All the adult stem cells, where do they all come from? An external source for organ-specific stem cell pools.

Stem cells can self-renew and maintain the ability to differentiate into mature lineages. Whereas the "stemness" of embryonic stem cells is not discussed, the primitiveness of a stem cell type within adult organisms is not well determined. Data presently available are either inconclusive or controversial regarding two main topics: maintenance or senescente of the adult stem cell pool; and pluripotentiality of the cells. While programmed senescence or apoptosis following uncorrected mutations represent no problem for mature cells, the maintenance of the stem cell pool itself must be assured. Two different mechanisms can be envisaged for that. In the first mechanism, which is generally accepted, stem cells originate during ontogeny along with the organ which they are responsible for, and remain there during all the lifespan of the organism. Several observations derived from recent reports allow the suggestion of a second mechanism. These observations include: organ-specific stem cells are senescent; adult stem cells circulate in the organism; stem cell niches are essential for the existence and function of stem cells; adult stem cells can present lineage markers; embryo-like, pluripotent stem cells are present in adult organisms, as shown by the development of teratomas, tumors composed of derivatives of the three germ layers; and the fact that the gonads may be a reservoir of embryo-like, pluripotent stem cells in adult organisms. The second mechanism for the maintenance of adult stem cells compartments implies a source external to the organ they belong, consisting of pluripotent, embryo-like cells of unrestricted life span, presenting efficient mechanisms for avoiding or correcting mutations and capable to circulate in the organism. According to this model, primitive stem cells exist in a specific organ in adult organisms. They undergo asymmetrical divisions, which originate one "true" stem cell and another one which enters the pool of adult stem cells, circulating through the entire organism. Upon signals liberated by organ-specific niches, this cell becomes activated to express lineage-specific genes, homes to that particular organ and repopulates its stem cell compartment, differentiating thus in what is seen as the organ-specific stem cell. The gonads are the natural candidates for homing the primitive stem cells in adult organisms. The model proposed in this work for the maintenance of organ-specific stem cell pools from an external source, represented by primitive, embryo-like germinal stem cells present in testes and ovaries, may contribute to the more complete understanding of this complex issue.

Adult↗
PubMedDate not supplied

[Specificity of the protein distribution in the nucleus and cytoplasm of the cells of provisional and definitive structures in early human embryos].

The highest amount of total protein in a 17-day human embryo is found in nuclei of the ectodermal germ layer. Histones are distributed as small clods whereas histones-like proteins vary in quantity in the apical and basal parts of the cells. During the 2nd and 3d months of embryogenesis protein topography in cells of different organs and systems is not the same and corresponds to the human histo- and organogenesis rate. Protein amount is especially high in chromosomes of dividing cells.

Cell Nucleus↗
PubMed2000

Regional specification during embryogenesis in the craniiform brachiopod Crania anomala.

A fate map has been constructed for the embryo of Crania. The animal half of the egg forms the ectodermal epithelium of the larva's apical lobe. The vegetal half of the egg forms endoderm, mesoderm, and the ectoderm of the mantle lobe. The vegetal pole is the site of gastrulation; this site becomes the posterior ventral region of the mantle lobe of the larva. The plane of the first cleavage goes through the animal-vegetal axis of the egg; it bears no relationship to the future plane of bilateral symmetry of the larva. The timing of regional specification was examined by isolating animal, vegetal, or meridional halves from oocytes, eggs, or embryos from prior to germinal vesicle breakdown through gastrulation. Animal halves isolated from oocytes formed either the epithelium of the apical lobe or a larva with all three germ layers. Animal halves isolated from unfertilized eggs and eight-cell embryos formed only apical lobe epithelium. Beginning at the blastula stage, animal halves formed mantle in addition to apical lobe epithelium. In animal halves isolated after gastrulation, the mantle lobe was always truncated. Vegetal halves isolated at all stages prior to gastrulation gastrulated and formed apical and mantle lobes with endoderm and mesoderm; however, the relative size of the apical lobe that formed decreased substantially when vegetal halves were isolated at later developmental stages. When meridional halves were isolated from unfertilized eggs and two- to four-cell embryos, both halves frequently formed normally proportioned larvae. Beginning at the blastula stage, a number of pairs frequently had a member that lacked dorsal setae on its mantle lobe while the other member of the pair formed setae, indicating that the dorsoventral axis had been set up. The process of regional specification in Crania is compared to those of Discinisca and Glottidia in the brachiopod subphylum Linguliformea and Phoronis in the phylum Phoronida.

Animals↗