Biomedical subjects

R A Pedersen

Publications and source records attributed to R A Pedersen.

At least 37 records · Page 2Linked to original sources

PubMed1998

FGF-2 alters the fate of mouse epiblast from ectoderm to mesoderm in vitro.

We have developed an in vitro differentiation assay to characterize the ability of peptide growth factors to induce differentiation in mouse epiblast. We report that culturing explants of mouse anterior epiblast, a tissue normally fated to give rise to neuroectoderm and surface ectoderm, in a serum-free, chemically defined medium with 10-50 ng/ml of FGF-2 induced gross changes in cell morphology. Treated cells adopted an elongated, flattened morphology but did not migrate from the explant. Instead, FGF-2-treated cells condensed into multicellular mounds or ridges. Immunocytochemistry showed that cells in treated explants expressed vimentin and in situ hybridization demonstrated that FGF-2 induced the expression of brachyury, goosecoid, and myo-D in regions of treated explants displaying morphological differentiation. Control explants cultured with platelet-derived growth factor AA (PDGF AA), transforming growth factor-beta 1 (TGF-beta 1), or in defined medium alone showed no morphological or biochemical differentiation. These results indicate that FGF-2 altered the fate of mouse anterior epiblast from ectoderm to mesoderm in vitro. Cell migration, which is characteristic of primitive streak mesoderm in vivo, was not induced by FGF-2 in these assays. However, the changes in morphology and the expression of mesodermal genes in vitro do support an early role for FGF signaling in the induction of mouse primitive streak mesoderm, as well as in later patterning events during embryogenesis.

Animals↗

PubMed1998

Spectral imaging in preconception/preimplantation genetic diagnosis of aneuploidy: multicolor, multichromosome screening of single cells.

PURPOSE: Our purpose was to evaluate the utility of spectral imaging for multicolor, multichromosome enumeration in human interphase cell nuclei. METHODS: Chromosome-specific probes labeled with different fluorochromes or nonfluorescent haptens were obtained commercially or prepared in-house. Metaphase spreads, interphase lymphocytes, or blastomeres cells were hybridized with either 7 or 11 distinctly different probes. Following 46 hr of hybridization, slides were washed and detected using either a filter-based quantitative image processing system (QUIPS) developed in-house or a commercial spectral imaging system. RESULTS: The filter-based fluorescence microscope system is preferred for simultaneous detection of up to seven chromosome targets because of its high sensitivity and speed. However, this approach may not be applicable to interphase cells when 11 or more targets need to be discriminated. Interferometer-based spectral imaging with a spectral resolution of approximately 10 nm allows labeling of chromosome-specific DNA probes with fluorochromes having greatly overlapping emission spectra. This leads to increases in the number of fluorochromes or fluorochrome combinations available to score unambiguously chromosomes in interphase nuclei. CONCLUSIONS: Spectral imaging provides a significant improvement over conventional filter-based microscope systems for enumeration of multiple chromosomes in interphase nuclei, although further technical development is necessary in its application to embryonic blastomeres. When applied to preconception/preimplantation genetic diagnosis, presently available probes for spectral imaging are expected to detect abnormalities responsible for 70-80% of spontaneous abortions caused by chromosomal trisomies.

Aneuploidy↗

PubMed1998

Culture of human embryos for studies on the derivation of human pluripotent cells: a preliminary investigation.

Several different culture conditions were evaluated for culturing grade 4 embryos (containing 2-4 blastomeres and with >50% fragmentation) 68 h after fertilization to the blastocyst stage. Embryos were co-cultured with buffalo rat liver (BRL) cells in Menezo's B2 medium with or without 10% v/v synthetic serum substitute (SSS), co-cultured with BRL cells in KSOM with or without 10% SSS, or cultured in KSOM with 100 nM heparin binding epidermal growth factor. The most consistent development was obtained when embryos were co-cultured with BRL cells in KSOM. Rates of development to the blastocyst stage were between 27% and 40%. After reaching the blastocyst stage, continued culture of these blastocysts was only possible in a medium without serum. In a serum-deprived medium cells attached and showed initial outgrowth, but did not survive passaging. Using another approach, inner cell masses (ICMs), isolated from blastocysts with high efficiency using immunosurgery, were able to attach to a feeder layer in the presence of serum. Some ICMs differentiated whereas others could be successfully passaged up to four times. The embryonic cells were morphologically different from murine embryonic stem cells. Instead of well-defined colonies, the human colonies were characterized by individual cells and colonies without defined borders.

Animals↗

PubMed1998

The mini-micro-epididymal sperm aspiration for sperm retrieval: a study of urological outcomes.

Epididymal sperm aspiration and in-vitro fertilization (IVF) with intracytoplasmic sperm injection is an established treatment for obstructive azoospermia. Sperm aspiration is performed with either an incision or percutaneously. To control costs, minimize morbidity and retain the advantages of both approaches, we developed a mini-incision technique for epididymal aspiration and here report sperm retrieval and procedure-related outcomes. Twenty-six consecutive patients with obstructive azoospermia underwent epididymal sperm retrieval through a 1 cm incision with local anaesthesia to provide spermatozoa for concurrent IVF cycles. The quality of retrieved spermatozoa, the quantity of spermatozoa cryopreserved as well as anaesthetic requirement, recovery time and patient satisfaction were evaluated. Fresh epididymal spermatozoa were retrieved in 25 of 26 (96%) patients. In one patient, testicular sperm extraction was necessary. Excess motile spermatozoa were cryopreserved in 24 of 26 (92%) patients; a mean total motile count of 4.8x10(6) motile spermatozoa were banked. The procedure was performed with 62% of patients receiving minimal i.v. sedation. Post-procedure recovery was rapid, with a median time to return to work of 2.0 days with a median of 2.0 pain pills taken. Procedure-related satisfaction was high. The mini-micro-epididymal sperm aspiration achieves the goals of reliable retrieval of abundant epididymal spermatozoa with a single, minimally morbid procedure. It appears to combine the advantages of the incision and percutaneous approaches.

Adult↗

PubMed1998

Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells.

The endocrine pancreas is organized into clusters of cells called islets of Langerhans comprising four well-defined cell types: alpha beta, delta and PP cells. While recent genetic studies indicate that islet development depends on the function of an integrated network of transcription factors, the specific roles of these factors in early cell-type specification and differentiation remain elusive. Nkx2.2 is a member of the mammalian NK2 homeobox transcription factor family that is expressed in the ventral CNS and the pancreas. Within the pancreas, we demonstrate that Nkx2.2 is expressed in alpha, beta and PP cells, but not in delta cells. In addition, we show that mice homozygous for a null mutation of Nkx2.2 develop severe hyperglycemia and die shortly after birth. Immunohistochemical analysis reveals that the mutant embryos lack insulin-producing beta cells and have fewer glucagon-producing alpha cells and PP cells. Remarkably, in the mutants there remains a large population of islet cells that do not produce any of the four endocrine hormones. These cells express some beta cell markers, such as islet amyloid polypeptide and Pdx1, but lack other definitive beta cell markers including glucose transporter 2 and Nkx6.1. We propose that Nkx2.2 is required for the final differentiation of pancreatic beta cells, and in its absence, beta cells are trapped in an incompletely differentiated state.

Animals↗

PubMed1997

Role of the Dlx homeobox genes in proximodistal patterning of the branchial arches: mutations of Dlx-1, Dlx-2, and Dlx-1 and -2 alter morphogenesis of proximal skeletal and soft tissue structures derived from the first and second arches.

The Dlx homeobox gene family is expressed in a complex pattern within the embryonic craniofacial ectoderm and ectomesenchyme. A previous study established that Dlx-2 is essential for development of proximal regions of the murine first and second branchial arches. Here we describe the craniofacial phenotype of mice with mutations in Dlx-1 and Dlx-1 and -2. The skeletal and soft tissue analyses of mice with Dlx-1 and Dlx-1 and -2 mutations provide additional evidence that the Dlx genes regulate proximodistal patterning of the branchial arches. This analysis also elucidates distinct and overlapping roles for Dlx-1 and Dlx-2 in craniofacial development. Furthermore, mice lacking both Dlx-1 and -2 have unique abnormalities, including the absence of maxillary molars. Dlx-1 and -2 are expressed in the proximal and distal first and second arches, yet only the proximal regions are abnormal. The nested expression patterns of Dlx-1, -2, -3, -5, and -6 provide evidence for a model that predicts the region-specific requirements for each gene. Finally, the Dlx-2 and Dlx-1 and -2 mutants have ectopic skull components that resemble bones and cartilages found in phylogenetically more primitive vertebrates.

Animals↗

PubMed1997

Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis.

We present genetic evidence that integrins regulate epithelial-mesenchymal interactions during organogenesis. Mice with a mutation in the alpha8 gene do not express the integrin alpha8 beta1 and exhibit profound deficits in kidney morphogenesis. In wild-type animals, inductive interactions between the ureteric epithelium and metanephric mesenchyme are essential for kidney morphogenesis. In alpha8 mutant homozygotes, growth and branching of the ureteric bud and recruitment of mesenchymal cells into epithelial structures are defective. Consistent with these phenotypes, alpha8 expression is induced in mesenchymal cells upon contact with the ureter. Since none of its previously identified ligands appears likely to mediate the essential functions of alpha8 beta1 in kidney morphogenesis, we have used an alpha8 beta1-alkaline phosphatase chimera to localize novel ligand(s) in the growing ureter. The distribution of these ligand(s) makes them strong candidates for regulators of kidney morphogenesis.

Alleles↗

PubMed1997

Dual blastomere analysis improves reliability of preimplantation trembler mouse diagnosis.

Dual blastomere biopsy and independent blastomere analysis dramatically improved preimplantation diagnostic reliability as confirmed by testing the remaining biopsied eight-cell mouse embryo. The autosomal dominant trembler mouse point mutation was selected as a model for human preimplantation diagnosis because: (1) single cell assay failure is predicted to be the highest when testing autosomal dominant mutations; (2) point mutations represent the most common of all mutation categories and the most demanding mutation to assay reliably; and (3) the trembler mouse point mutation in peripheral myelin protein 22 (Pmp22) is a model of human Charcot-Marie-Tooth type 1A disease. Mathematical models predict our experimental results assuming amplification of 80% of each target allele as well as trembler sperm DNA contamination in 1 of 44 normal biopsied single blastomeres. Single blastomere analysis correctly predicted the genotype in only 84% of embryos that would have been implanted as normal. In contrast, when independent tests of both biopsied blastomeres agreed, test results were confirmed in 20 of 21 (95.2%) of the remaining six-cell biopsied embryos designated as normal. Thus, biopsied six-cell embryo confirmation demonstrated that dual biopsied blastomere analysis improved test reliability remarkably.

Animals↗

PubMed1997

Development in vitro of Marsupials: a comparative review of species and a timetable of cleavage and early blastocyst stages of development in Monodelphis domestica.

The development of marsupial oocytes and embryos in vitro is reviewed. Most stages of development have been cultured successfully, usually in a complex medium with added fetal calf serum. Simpler media without added serum have been developed for fertilization and cleavage in vitro. Culture systems have been established for oocyte maturation and fertilization in the grey short-tailed opossum and for cleavage from the zygote to the early expanding unilaminar blastocyst in a number of other marsupials. Survival in vitro of the unilaminar and early bilaminar blastocyst stages is limited in all species examined. In contrast, late bilaminar, trilaminar, embryonic and fetal stages develop at rates approximating those in vivo. More stages have been cultured successfully in Sminthopsis macroura than in any other species. It has been cultured from the late bilaminar blastocyst to within 18 h of birth. Stages of cleavage and unilaminar blastocyst formation of Monodelphis domestica timed by videotaping mating animals, proceeded at similar rates in vivo and in vitro. As in other marsupials, cleavage in this opossum is characterized by a polarized conceptus. This polarity is expressed in the distribution of organelles in the zygote and the localization of secretion of the extracellular matrix material into the cleavage cavity and of the initial cell-zona attachment. Because cell-cell adhesion follows cell-zona adhesion, a unilaminar blastocyst forms without the development of an intervening morula stage.

Animals↗

PubMed1996

Elevated DNA excision repair capacity in the extraembryonic mesoderm of the midgestation mouse embryo.

In order to determine whether there is differential cell-type-specific DNA repair we measured the nucleotide excision repair capacity of the four distinct cell lineages that comprise the extraembryonic yolk sac using the unscheduled DNA synthesis assay. Yolk sacs from mouse embryos at 11.5-12.5 days gestation were microdissected to yield purified trophoblast, parietal endoderm, mesoderm, and visceral endoderm, as well as fetal skin fibroblasts which were then grown as primary explants. At this midgestational stage of development, the yolk sac provides essential functions for the sustenance of the embryo while the complex process of organogenesis is proceeding in the liver, kidney, and gut. Trophoblast giant cells, parietal endoderm, and visceral endoderm all demonstrated low levels of unscheduled DNA synthesis consistent with levels measured in adult mouse skin fibroblasts. As has previously been documented, embryonic mouse skin fibroblasts were reproducibly 2- to 3-fold higher than adult mouse skin fibroblasts in levels of DNA excision repair. The extraembryonic mesoderm, however, displayed a statistically significant level of unscheduled DNA synthesis 10-fold higher than adult mouse skin fibroblasts or the other lineages of the midgestation yolk sac. Further, the S-indexes of these lineages were also determined to assess the possible relevance of differential repair to the proliferative status of the cells. These data demonstrate that DNA excision repair capacity is lineage-specific during embryogenesis in the mouse. These studies may begin to provide a context for understanding the perplexing developmental aspects such as the characteristic congenital abnormalities associated with the human heritable DNA repair deficiency diseases.

Animals↗

PubMed1996

Mutation of the Emx-1 homeobox gene disrupts the corpus callosum.

Expression of the Emx-1 homeobox gene is largely restricted to the developing and mature cerebral cortex. To study its function, two lines of mice were generated using gene targeting methods that have a deletion that includes the N-terminal coding region of Emx-1. Mice homozygous for the deletion were viable and fertile and exhibited no obvious behavioral defects. However, 100% of homozygous mice lack most or all of their corpus callosum, the principle fiber tract that connects the left and right cerebral hemispheres. Heterozygotes show partial penetrance for the corpus callosum abnormality. The histology and various molecular properties of the cerebral cortex appear normal in the mutant mice.

Agenesis of Corpus Callosum↗

PubMed1996

Defective chorioallantoic fusion in mid-gestation lethality of parthenogenone<-->tetraploid chimeras.

Previous studies of parthenogenetic embryos revealed severe perturbations of both embryonic and extraembryonic tissue lineages during postimplantation development. The majority of pure parthenogenetic concepti have no recognizable axis and exhibit preferential terminal differentiation of their trophectoderm and primitive endoderm. To further define the role of the extraembryonic lineages in parthenogenetic development, we provided them with zygote-derived extraembryonic tissues by aggregating them with fertilized tetraploid embryos. On Day 12 of combined in vitro and in vivo development, most of the embryos proper in these chimeras were entirely derived parthenogenetically, whereas their trophectoderm and primitive endoderm tissues were derived from the tetraploid component. No Igf2 expression was detected in the parthenogenetic embryo proper, indicating that imprinting was manifested in such chimeras. Typical development of the parthenogenetic embryo proper was markedly improved in comparison with pure parthenogenetic concepti, with such chimeras attaining an average of 23 somites (range, 10 to 35). However, most of the chimeras died abruptly at Day 13, and all were being resorbed at Day 14 of development. The gross normality of axial structures and organ development suggests that a major cause of failure of these chimeric parthenogenones to survive beyond mid-gestation was due to defective chorioallantoic fusion. Our results indicate that the severe perturbation of axial development seen in most pure parthenogenetic concepti is a secondary consequence of the effects of parthenogenesis on the trophectoderm and primitive endoderm lineages. Moreover, the mid-gestation death of parthenogenetic embryos proper despite the presence of zygote-derived tetraploid tissues implicates extraembryonic mesoderm in manifesting the effects of genomic imprinting.

Allantois↗

PubMed1996

Induction and differentiation of extra-embryonic mesoderm in the mouse.

Extra-embryonic mesoderm, derived at the time of gastrulation from the primitive streak, gives rise to several tissues that function to provide the embryo with nutrients, a means of waste disposal, and mechanical protection. Little is known about the differentiation of this tissue and about the growth and transcription factors involved. The present review focussed on growth and transcription factors that may be involved in differentiation of extra-embryonic mesoderm, and results from fate-mapping and transplantation studies. Methods in vitro available for assaying the effects of growth and transcription factors on development of extra-embryonic development are also discussed.

Animals↗

PubMed1996

Parthenogenetic activation of mouse oocytes using calcium ionophores and protein kinase C stimulators.

Fertilization involves the production of inositol trisphosphate and diacylglycerol with a subsequent increase in intracellular calcium concentration ([Ca2+]i) and the activation of a calcium-dependent protein kinase, the so-called protein kinase C (PKC). Methods of parthenogenetic activation have focused on this calcium wave which seems to be large enough to generate all the responses associated with fertilization and even finally inducing the activation of PKC activity. The specific stimulation of PKC by phorbol esters in turn elicits [Ca2+]i oscillations although no reports exist claiming that the mere activation of this protein is capable of sustaining embryonic development. In this paper we describe the effect of different calcium ionophores and phorbol esters as parthenogenetic agents on mouse oocytes compared with ethanol as the standard procedure. Phorbol esters (OAG) fail to activate a significant number of oocytes, with very few reaching blastocyst stage. However, when a calcium ionophore (A23187) is added, the percentage of embryos reaching the blastocyst stage increases to such an extent that it is the best chemical method assayed to date. We conclude that incubation with both compounds combined inhibits feed-back processes between the above reactions and so induces a more physiologic parthenogenetic activation.

Animals↗

PubMed1995

Null mutation of Dlx-2 results in abnormal morphogenesis of proximal first and second branchial arch derivatives and abnormal differentiation in the forebrain.

Genetic analysis of the development and evolution of the vertebrate head is at a primitive stage. Many homeo box genes, including the Distal-less family, are potential regulators of head development. To determine the function of Dlx-2, we generated a null mutation in mice using gene targeting. In homozygous mutants, differentiation within the forebrain is abnormal and the fate of a subset of cranial neural crest cells is respecified. The latter causes abnormal morphogenesis of the skeletal elements derived from the proximal parts of the first and second branchial arches. We hypothesize that the affected skull bones from the first arch have undergone a transformation into structures similar to those found in reptiles. These results show that Dlx-2 controls development of the branchial arches and the forebrain and suggests its role in craniofacial evolution.

Animals↗

PubMed1995

Novel flow-cytometric method for separating cell types in differentiated F9 embryoid bodies.

The differentiation of F9 teratocarcinoma cells mimics the formation of a mouse embryonic tissue, the primitive endoderm. In vitro, small aggregates of F9 cells, termed embryoid bodies, differentiate in response to retinoic acid and develop a surface epithelium that is characterized by the production of alpha-fetoprotein. In the present study, cellular autofluorescence profiles obtained by fluorescence-activated embryoid bodies were composed of a single type of cell. In contrast, retinoic acid-induced embryoid bodies were composed of two cell types: a major population displaying autofluorescence levels similar to those of cells from undifferentiated embryoid bodies and a second population displaying higher autofluorescence. RNA analyses demonstrated that the transcription of alpha-fetoprotein was associated only with the more highly autofluorescent population, indicating that flow cytometry provides a novel mechanism for the separation of undifferentiated cells from differentiated endoderm cells in F9 embryoid bodies.

Animals↗

PubMed1995

Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality.

Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when beta 1 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the beta 1 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of beta 1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking beta 1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 beta 1-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 beta 1-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in beta 1-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin- and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of beta 1-null blastocyst outgrowths, and inner cell masses isolated from beta 1-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that beta 1 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in beta 1-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for beta 1 integrins for later stages of trophoblast development, or to the lack of trophic signals from the beta 1-null inner cell mass.

Animals↗

PubMed1995

Epithelial immaturity and multiorgan failure in mice lacking epidermal growth factor receptor.

Since the discovery that epidermal growth factor (EGF) can accelerate opening of the eyelids, the EGF receptor (EGF-R) has been extensively studied and is now considered to be a prototype tyrosine kinase receptor. Binding of EGF or of transforming growth factor-alpha (TGF-alpha) or other related factors activates the receptor and induces cell proliferation and differentiation. Although it is not found on haematopoietic cells, the EGF-R is widely expressed in mammals and has been implicated in various stages of embryonic development. Here we investigate the developmental and physiological roles of this receptor and its ligands by inactivating the gene encoding EGF-R. We find that EGF-R-/- mice survive for up to 8 days after birth and suffer from impaired epithelial development in several organs, including skin, lung and gastrointestinal tract.

Animals↗