Biomedical subjects

R A Pedersen

Publications and source records attributed to R A Pedersen.

At least 55 records · Page 3Linked to original sources

PubMed1995

Allocation of epiblast cells to germ layer derivatives during mouse gastrulation as studied with a retroviral vector.

The embryonic ectoderm, or epiblast, is the source of the three primary germ layers that form during gastrulation in the mouse embryo. Previous studies have investigated the fate of epiblast cells in early gastrulation stages using clonal analysis of cell lineage and in late gastrulation stages using transplantation of labeled grafts. In this study, we studied the fate of late gastrulation stage epiblast using a clonal analysis based on a retroviral vector encoding the Escherichia coli lacZ gene. We found that by reducing the volume of viral suspension injected into each embryo, it was possible to achieve single infectious events. Our analysis of 20 embryos singly infected at the late streak stage and 21 at the head fold stage revealed clonal descendants in only a single germ layer in each embryo. These results indicate that allocation of epiblast progenitors to a single germ layer fate has occurred by late gastrulation in mouse embryos.

Animals↗

PubMed1995

Developmental regulation of genomic imprinting during gametogenesis.

Successful mammalian development requires both the male and female genomes. This is due in part to genomic imprinting, which results in offspring inheriting only one functional copy of a gene from either the mother or the father. Evidence suggests that this specialization of the parental genomes is established during gametogenesis when the imprint pattern inherited from the parent is switched to reflect the sex of the progeny. We used reverse transcription-PCR to analyze the allele-specific expression of Igf-2, Igf-2r, and H19 in the testes and ovaries of mice derived from an interspecies cross between Mus musculus and Mus spretus. Because of genomic imprinting, Igf-2 is expressed only from the paternal allele and Igf-2r and H19 only from the maternal allele, in most tissues. Although allele-specific expression was maintained in the neonatal testis and ovary, relaxation of imprinting was detected by 7 days after birth in the male and continued during testis development. In the female, relaxation of the Igf-2 and Igf-2r parental imprints was observed in the adult ovary and oocyte. These results (1) indicate that imprinted expression is relaxed during gametogenesis, presumably as a consequence or prerequisite of the imprinting mechanism, and (2) predict a subsequent imprinting event after which the allele-specific expression of Igf-2, Igf-2r, and H19 reflects the parent of origin.

Alleles↗

PubMed1994

Abnormal development of embryonic and extraembryonic cell lineages in parthenogenetic mouse embryos.

Parthenogetically activated, diploid mouse oocytes can develop to midgestation stages in utero. However, even these advanced parthenogenones appear to die because of much reduced trophoblast and yolk sac development. Previous studies have compared the general features of parthenogenetic and androgenetic development and determined the fate of uniparental cells in chimeras with normal embryos. These studies led to the concept of genomic imprinting as the cause for developmental failure when either the maternal or the paternal genome is duplicated, with the corresponding deficiency of the other. Genomic imprinting appears to arise during gametogenesis and to act through dosage effects in a set of imprinted genes, whose expression depends on their parental origin. In this study we undertook a more detailed morphological analysis of parthenogenetic development in the mouse and established a classification system to quantify the developmental extent of parthenogenones. We found that the failure of parthenogenones occurred at different times during early postimplantation development, generating a spectrum of concepti which had developed to different extents, with only a small fraction of the embryos reaching advanced somite stages. In all parthenogenones differentiation and proliferation of the trophectoderm and primitive endoderm lineages (both extraembryonic) was abnormal, and in all, even the best-developed parthenogenones, we observed similar deficiencies in the embryonic lineages, especially the mesoderm. Common to all abnormally developed lineages was that the proportion of undifferentiated precursor cells was much reduced, while their differentiated descendants were relatively abundant. We propose, therefore, that the failure of parthenogenones to develop to term is due to abnormal regulation of differentiation and proliferation in both embryonic and extraembryonic lineages. In this hypothesis, the apparent tissue specific defects observed in parthenogenones arise as a consequence of the functional importance of certain tissues (like the trophoblast) early in development. The spectrum of parthenogenones thus appears to reflect critical events in early development, whose regulation are affected by genomic imprinting.

Animals↗

PubMed1994

Spatially restricted imprinting of mouse chromosome 7.

Three of the four known imprinted genes (Igf-2, H19, and Snrpn) map to mouse chromosome 7. We used mRNA phenotyping to examine the tissue-specific transcription of Igf-1r, H-ras-1, and Gabrb3, which map to chromosome 7 between Snrpn and the Igf-2/H19 domain, and Myod-1, which maps proximal to Snrpn. We found that all of these genes were expressed by both parental alleles in tissues from day 1 neonates. The fact that imprinted genes can flank or map closely to genes that escape such epigenetic modification suggests that autosomal imprinting is not manifested globally along imprinted chromosomes but rather is spatially restricted, perhaps even defined by specific DNA consensus sequences or an "imprint box" associated with imprintable genes.

Alleles↗

PubMed1994

Timetable of in vivo embryonic development in the grey short-tailed opossum (Monodelphis domestica).

The timing of development was examined in 496 embryos from female Monodelphis domestica collected at known time intervals after video recorded mating. Ovulation occurred approximately 20 hr (day 1) after mating, and fertilization was observed by 24 hr. Transport through the oviducts was rapid, and pronuclear stage embryos were recovered from the uterus as early as 24 hr after mating. Second cleavage had occurred by 55 hr after mating. Three-celled embryos were among those collected on day 3 after mating, indicating that asynchronous cleavage of blastomeres can occur from the two-cell stage. The four-cell stage persisted for approximately 24 hr, and embryos that had undergone third cleavage were first recovered 74 hr after mating. Embryos that had undergone fourth to fifth cleavage were found 96-100 hr (4 days) after mating and complete unilaminar blastocysts by 5.5 days after mating. Primary endoderm formed from an already distinct embryonic area of the unilaminar blastocyst early on day 7 after mating. Formation of the bilaminar blastocyst was completed rapidly, on day 7 after mating. The primitive streak appeared on day 10 after mating, and organogenesis rapidly ensued on a timetable similar to that reported for Didelphis virginiana (McCrady, 1938). Close contact with the maternal circulation was established on day 11 and by day 12 maternal and embryonic tissues could not be separated without damage. The length of the gestation period from fertilization to birth was approximately 13.5 days. These observations provide the basis for further embryological cellular and molecular studies of this species as a laboratory model for marsupial development.

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PubMed1994

Mechanisms of genomic imprinting in mammals.

This chapter can be summarized by the following main points: Genomic imprinting results in the functional nonequivalence of the maternal and paternal genomes, thereby preventing the development of viable parthenogenotes and androgenotes in eutherian mammals. Imprinting may have arisen as a result of the specialized evolutionary requirements of the parental genomes or may have been an obligatory step in the development of placentation. A substantial proportion of transgenes and a smaller number of endogenous genes demonstrate imprinted pattern of expression in mice and humans. An analysis of DNA methylation in somatic tissues and germ cells during embryonic and postnatal development reveals dynamic changes, particularly during gametogenesis and early embryogenesis. The nature and timing of these changes suggest that DNA methylation may be involved in genomic imprinting. Imprinted genes display complex methylation patterns. Many aspects of these patterns are consistent with a role for methylation in the imprinted phenotype, although it is currently unclear whether methylation functions in the establishment of imprinting or plays a secondary role in the maintenance of the imprinted pattern of expression. Studies underway to identify new imprinted genes may help elucidate both the function and mechanism of genomic imprinting.

5-Methylcytosine↗

PubMed1994

Parental imprinting of the Mas protooncogene in mouse.

The Mas protooncogene on mouse chromosome 17 encodes a mitogenic G-protein-coupled cell surface receptor. We investigated the allele-specific expression pattern of the Mas gene on the basis of its proximity to the known imprinted gene for the insulin growth factor type II receptor (Igf2r). Phenotyping of mRNA demonstrated exclusive expression from the paternal allele in all embryonic tissues, including visceral yolk sac, between 11 and 12.5 days of gestation. By 13.5 days of gestation the paternal allele-specific expression of Mas was restricted to heart, tongue and visceral yolk sac, whereas all other tissues exhibited relaxation of the parental imprint. Our results demonstrate parental imprinting of Mas and suggest that the maternally inherited allele is transcriptionally repressed in a developmental and tissue-specific manner.

Animals↗

PubMed1994

Studies of in vitro differentiation with embryonic stem cells.

Embryonic stem (ES) cells were first cultured from mouse embryos little more than a decade ago, yet they are now widely used in transgenic studies which are revolutionizing mammalian genetics. Although drawing less attention, in vitro studies of mouse ES cells have also contributed widely to the understanding of mechanisms of embryonic cell differentiation and proliferation. This review focusses on the application of ES cells as in vitro models for cellular and molecular events in the early mammalian embryo. Future studies with cultured ES cells of mouse and other species should provide insights into the factors regulating the differentiation of intermediate stem cells and terminal cells for the various embryonic lineages, thus contributing profoundly to the understanding of mammalian embryogenesis as well as providing cells for therapeutic applications.

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PubMed1993

Embryonic expression of human keratin 18 and K18-beta-galactosidase fusion genes in transgenic mice.

During embryogenesis, EndoB, the mouse form of human keratin 18 (K18), is expressed in a complex spatial and temporal pattern in various embryonic epithelia. We have compared the expression of transgenic human K18 to the endogenous mouse homolog and to the coexpressed, complementary keratin 8 homolog, EndoA, during postimplantation mouse embryogenesis and fetal development in order to determine the developmental expression pattern of the human gene in a mouse environment. The tissue distribution of K18 protein was identical to that of endogenous EndoB in both 7.5- and 13.5-day-old embryos, except for certain heart, eye, and extraembryonic mesodermal tissues in which K18 was not detected. These results indicate that the 10-kb K18 gene specifies appropriate developmental expression in the mouse and support previously reported differences in K18 expression in human and mouse fetal heart. We have also compared the expression patterns of K18 to a series of constructions that utilize the Escherichia coli gene for beta-galactosidase (lacZ) as a reporter gene. Some of these constructions were regulated correctly in embryos during development of the germ layers. However, none was expressed consistently in extraembryonic or in adult tissues. Analysis with methylation-sensitive restriction enzymes revealed that hypermethylation of the CpG-rich prokaryotic reporter gene was not the cause of its silence in adult transgenic liver. However, the repressed state of K18-LacZ transgenes in adult liver was correlated with a different chromatin state that lacked diagnostic DNase hypersensitive sites found in K18 transgenic liver. Expression of the lacZ reporter gene did not accurately reflect the developmental pattern of K18 even in constructions that used all available K18 sequences. We conclude that in these contexts, the lacZ gene was not a developmentally neutral reporter gene.

Animals↗

PubMed1993

Glucose-dependent insulinotropic polypeptide stimulated insulin release from a tumor-derived beta-cell line (beta TC3).

The beta TC3 tumor cell line was examined for the presence of functional glucose-dependent insulinotropic polypeptide (GIP) receptors. Increasing amounts of natural porcine GIP decreased the binding of HPLC-purified [125I]GIP to beta TC3 cells in a concentration-dependent manner. Displacement of GIP was significant at concentrations as low as 500 pM, and the radioligand was fully displaced at 100 nM. GIP(1-30) produced a displacement of [125I]GIP comparable with that produced by GIP(1-42), and glucagon yielded 20% displacement at a concentration of 1 microM but was without effect at 100 mM. Incubation of beta TC3 cells in the presence of glucose concentrations of 2-20 mM yielded a concentration-dependent stimulation of immunoreactive insulin (IRI) release. GIP and glucagon-like peptide-I(7-36) amide (tGLP-I) at concentrations of 1 nM or greater significantly stimulated IRI release in the presence of 2 mM glucose. The threshold glucose concentration for GIP-stimulated IRI release from beta TC3 cells was 0.5 mM, and maximal potentiation of IRI release by GIP occurred at 5 mM glucose. Somatostatin significantly inhibited GIP-stimulated IRI release in the presence of 5 mM glucose. It is concluded that beta TC3 cells have functional GIP receptors and may provide a useful model for the study of IRI secretion at the cellular level.

Animals↗

PubMed1993

The role of E-cadherin and integrins in mesoderm differentiation and migration at the mammalian primitive streak.

We have examined the role of cell-cell and cell-extracellular matrix (ECM) interactions during mesoderm differentiation and migration at the primitive streak of the mouse embryo with the use of function-perturbing antibodies. Explants of epiblast or mesoderm tissue dissected from the primitive streak of 7.5- to 7.8-day mouse embryos were cultured on a fibronectin substratum in serum-free, chemically defined medium. After 16-24 hours in culture, cells in explants of epiblast exhibited the typical close-packed morphology of epithelia, and the tissue remained as a coherent patch of cells that were shown to express transcripts of the cytokeratin Endo B by in situ analysis. In contrast, cells in explants of primitive streak mesoderm exhibited a greatly flattened, fibroblastic morphology, did not express Endo B transcripts, and migrated away from the center of the explant. As epiblast cells in vivo undergo the epithelial-mesenchymal transition at the primitive streak, they cease expressing the prominent calcium-sensitive cell adhesion molecule E-cadherin (uvomorulin, Cell-CAM 120/80). We asked whether the loss of E-cadherin expression was a passive result of differentiation or if it might play a more causative role in mesoderm differentiation and migration. Culture with function-perturbing antibodies against E-cadherin caused cells within epiblast explants to lose cell-cell contacts, to flatten, and to assume a mesenchymal morphology; they were also induced to migrate. Anti-E-cadherin antibodies had no effect on explants of primitive streak mesoderm. In immunofluorescence studies, anti-E-cadherin-treated epiblast cells ceased to express SSEA-1, a carbohydrate moiety that is lost as mesoderm differentiates from the epiblast in vivo, and they also ceased to express E-cadherin itself. In contrast, these cells began to express the intermediate filament protein vimentin, a cytoskeletal protein characteristic of the primitive streak mesoderm at this stage of development. As epiblast cells differentiate into mesoderm, their predominant adhesive interactions change from cell-cell to cell-substratum. Therefore, we also investigated the adhesive interactions between primitive streak tissues and extracellular matrix (ECM) components. Epiblast explants adhered well to fibronectin, more poorly to laminin and type IV collagen, and not at all to vitronectin. In contrast, mesoderm explants attached well to all these proteins. Furthermore, epiblast, but not mesoderm, displayed an anchorage-dependent viability in culture. After anti-E-cadherin treatment, epiblast cells that had assumed the mesenchymal morphology did attach to vitronectin, another characteristic shared with primitive streak mesoderm.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Preimplantation diagnosis.

Preimplantation embryonic biopsy and analysis offer couples at increased risk of having offspring affected with a genetic disorder the possibility of an early prenatal diagnosis. For many couples, this approach would avoid the issue of the selective termination of affected fetuses. Substantial advances have been made in the area of preimplantation diagnosis, but the possible difficulties with this approach cannot be ignored.

Animals↗

PubMed1992

Clonal analysis of cell fate during gastrulation and early neurulation in the mouse.

The foundation of the germ layers and the extraembryonic mesoderm from the epiblast between 6.5 and 7.5 days post coitum (p.c.) is accompanied by substantial cell proliferation. It is followed during the next 24 hours by the organization of major systems of the embryo such as the central nervous system, somites, heart and vascular system. Injection in situ of a short-term lineage label (horse radish peroxidase) into single epiblast cells at 6.7 days p.c. and analysis of the descendant clones in cultured embryos have been used to trace these processes and led to the following conclusions: (1) There is extensive but not indiscriminate cell mixing at the onset of gastrulation; epiblast cells spread towards the primitive streak and descendants are there progressively incorporated into mesoderm. (2) The fate map of the mouse epiblast at the early primitive streak stage is topologically similar to those of other vertebrates. (3) Germ layers and the extraembryonic mesoderm are not clonally distinct before gastrulation, the region of overlapping boundaries in the fate map being occupied by cells that will have descendants in more than one layer. (4) Cranial neurectoderm is mainly derived from axial epiblast immediately anterior to the primitive streak of the early streak stage embryo, clonal descendants being spread rostrocaudally in the developing neural tube. Contribution to the putative floor plate is made by progenitors some of which also contribute to notochord and mesoderm.

Animals↗

PubMed1992

Parent-specific expression of a human keratin 18/beta-galactosidase fusion gene in transgenic mice.

Insertion of a human keratin 18 (K18)-bacterial beta-galactosidase (LacZ) fusion gene into mice has led to a unique transgenic line in which expression of the transgene is subject to unusual germ line-specific, genomic imprinting effects. Fetal expression of the LacZ reporter gene depends on the gender of the transmitting parent, with appropriate expression in liver after maternal inheritance, and ectopic expression in retina and mesodermal tissues after paternal inheritance. This tissue-specific imprinting pattern is superimposed upon a basic expression pattern which is unaffected by parental inheritance. Insertion of the transgene has led to a recessive-lethal phenotype, with no parent-of-origin effects on viability, suggesting that the transgene has not inserted into an imprinted region of the genome. HpaII and HhaI methylation sensitive restriction sites within the bacterial LacZ reporter gene are completely methylated when activity of the maternally inherited transgene is detected in the fetal liver, and not methylated when the paternally inherited transgene is silent. Thus DNA methylation of LacZ is correlated with maternal inheritance and may be implicated in the genomic imprinting mechanism as others have suggested. However, in contrast to the commonly found correlation of expression and low DNA methylation, the LacZ gene was expressed in fetal liver when fully methylated. This result may imply the existence of negative regulatory activities that recognize the unmethylated LacZ gene.

Animals↗

PubMed1992

Insulin-like growth factor II acts through an endogenous growth pathway regulated by imprinting in early mouse embryos.

We present evidence that insulin-like growth factor II (IGF-II) mediates growth in early mouse embryos and forms a pathway in which imprinted genes influence development during preimplantation stages. mRNA and protein for IGF-II were expressed in preimplantation mouse embryos, but the related factors IGF-I and insulin were not. IGF-I and insulin receptors and the IGF-II/mannose-6-phosphate receptor were expressed. Exogenous IGF-II or IGF-I increased the cell number in cultured blastocysts, but a mutant form of IGF-II that strongly binds only the IGF-II receptor did not. Reduction of IGF-II expression by antisense IGF-II oligonucleotides decreased the rate of progression to the blastocyst stage and decreased the cell number in blastocysts. Preimplantation parthenogenetic mouse embryos expressed mRNA for the IGF-II receptor but not for either IGF-II ligand or the IGF-I receptor, indicating that the latter genes are not expressed when inherited maternally. These data imply that some growth factors and receptors, regulated by genomic imprinting, may control cell proliferation from the earliest stages of embryonic development.

Animals↗

PubMed1991

Drosophila alcohol dehydrogenase: a novel reporter gene for use in mammalian embryos.

A reporter gene construct containing the Drosophila alcohol dehydrogenase (ADH) gene under regulation of the Rous sarcoma virus long terminal repeat (RSV LTR) was microinjected into mouse zygote pronuclei. ADH activity after injection of the RSV-ADH construct was visualized in cultured embryos by means of a simple histochemical enzyme assay. The RSV LTR was an efficient promoter that led to abundant ADH activity at all stages of preimplantation development. There was a high incidence of mosaicism among stained embryos.

Alcohol Dehydrogenase↗

PubMed1991

Induction of c-fos transcripts in early postimplantation mouse embryos by TGF-alpha, EGF, PDGF, and FGF.

The activity of growth factor receptors in the early postimplantation mouse embryo was studied by analyzing changes in expression of mRNA transcripts of an early response gene, c-fos, after binding of specific ligands. Reverse transcription of mRNA coupled with the polymerase chain reaction was used to detect gene transcription in single embryos after exposure to growth factors. Postimplantation embryos (at 7.5 days of gestation) had physiologically active receptors for transforming growth factor-alpha (TGF-alpha), epidermal growth factor (EGF), human platelet-derived growth factor (PDGF), recombinant PDGF-AA homodimer, and basic fibroblast growth factor (FGF), as indicated by induced expression of c-fos mRNA, c-fos expression was not induced in untreated embryos or in embryos incubated with active recombinant PDGF-BB homodimer. These results show that growth factor receptors are functional during early mammalian embryogenesis.

Animals↗

PubMed1991

Clonal analysis of epiblast fate during germ layer formation in the mouse embryo.

The fate of cells in the epiblast at prestreak and early primitive streak stages has been studied by injecting horseradish peroxidase (HRP) into single cells in situ of 6.7-day mouse embryos and identifying the labelled descendants at midstreak to neural plate stages after one day of culture. Ectoderm was composed of descendants of epiblast progenitors that had been located in the embryonic axis anterior to the primitive streak. Embryonic mesoderm was derived from all areas of the epiblast except the distal tip and the adjacent region anterior to it: the most anterior mesoderm cells originated posteriorly, traversing the primitive streak early; labelled cells in the posterior part of the streak at the neural plate stage were derived from extreme anterior axial and paraxial epiblast progenitors; head process cells were derived from epiblast at or near the anterior end of the primitive streak. Endoderm descendants were most frequently derived from a region that included, but extended beyond, the region producing the head process: descendants of epiblast were present in endoderm by the midstreak stage, as well as at later stages. Yolk sac and amnion mesoderm developed from posterolateral and posterior epiblast. The resulting fate map is essentially the same as those of the chick and urodele and indicates that, despite geometrical differences, topological fate relationships are conserved among these vertebrates. Clonal descendants were not necessarily confined to a single germ layer or to extraembryonic mesoderm, indicating that these lineages are not separated at the beginning of gastrulation. The embryonic axis lengthened up to the neural plate stage by (1) elongation of the primitive streak through progressive incorporation of the expanding lateral and initially more anterior regions of epiblast and, (2) expansion of the region of epiblast immediately cranial to the anterior end of the primitive streak. The population doubling time of labelled cells was 7.5 h; a calculated 43% were in, or had completed, a 4th cell cycle, and no statistically significant regional differences in the number of descendants were found. This clonal analysis also showed that (1) growth in the epiblast was noncoherent and in most regions anisotropic and directed towards the primitive streak and (2) the midline did not act as a barrier to clonal spread, either in the epiblast in the anterior half of the axis or in the primitive streak. These results taken together with the fate map indicate that, while individual cells in the epiblast sheet behave independently with respect to their neighbours, morphogenetic movement during germ layer formation is coordinated in the population as a whole.

Animals↗