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Biomedical subjects

R L Gardner

Publications and source records attributed to R L Gardner.

At least 37 records · Page 2Linked to original sources
PubMed1999

Polarity in early mammalian development.

Although overall polarity is discernable morphologically in both the growing and mature oocyte in mammals, it is typically relatively inconspicuous in the zygote. Furthermore, the conceptus exhibits an essentially radial organization during cleavage which was long held to persist until the primitive streak forms at the onset of gastrulation. This view has been challenged by various recent studies which clearly show that asymmetries are evident both morphologically and at the molecular level from very early in development. Collectively, these new findings argue that specification of the anterior-posterior axis of the fetus depends on information that is localized extra-embryonically in cells which begin to differentiate before the conceptus implants in the uterus.

Animals↗
PubMed1999

Alzheimer's disease: correlation of the suppression of beta-amyloid peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome.

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.

Aldehydes↗
PubMed1999

Insulin-like growth factor-2 regulation of conceptus composition: effects of the trophectoderm and inner cell mass genotypes in the mouse.

The purpose of this study was to measure the effect of insulin-like growth factor-2 deficiency on the growth of the mouse conceptus. Initial observations on normal development in the 129J/Sv strain established that wet and dry weights were reduced by 35% when the insulin-like growth factor-2 gene was inactive. The DNA contents were reduced by only 15%. We exchanged the inner cell mass and trophectoderm between mouse blastocysts that had or lacked an active insulin-like growth factor-2 gene. At embryonic Day 16.5, lack of this gene's activity in the derivatives of either tissue decreased the fluid volumes of the exocelomic and amniotic cavities. The wet weights of the "fetal placentas," the yolk sacs, and the fetuses were also decreased. However, the tissue wet weight decrease could not be accounted for by the change in DNA content, indicating that cell-associated biomass had changed. The conclusions are 1) that insulin-like growth factor-2 levels regulate the composition of the fetus and extra-embryonic tissues and 2) that trophectoderm and inner cell mass derivatives cooperate to control extra-cellular fluid volume in the conceptus.

Amnion↗
PubMed1998

Contributions of blastocyst micromanipulation to the study of mammalian development.

This is a personal account of why the author chose to focus on devising techniques for micromanipulating the blastocyst stage conceptus as a way of investigating early development in mammals. Its aim is to provide insight into what such technical innovations entailed and how they have contributed to present understanding of both embryology and the analysis of gene function in mammals. The ability to dissect and reconstitute mouse blastocysts, and to inject cells or tissue into them, enabled genes to be harnessed as markers for elucidating the lineage of cells and interactions between tissues from the stage when differentiation is first evident. Most importantly, it made it possible to apply clonal analysis to the study of cell fate in mammals. The scope of blastocyst micromanipulation was further enhanced when embryonal carcinoma (EC) cells and, particularly, embryonic stem (ES) cells were found to be able to participate in normal development and contribute to the germ line following injection into the blastocyst.

Animals↗
PubMed1998

Translational inhibition of cyclin B1 and appearance of cyclin D1 very early in the differentiation of mouse trophoblast giant cells.

Investigation of the basis of uncoupling of replication of the genome from mitosis in the mouse trophoblast has so far been neglected despite its significance for understanding both placental development and cell cycle control. In order to obtain clues about the molecular basis of the switch from proliferation to endoreduplication, we have investigated changes in the expression of cyclins and cyclin-dependent kinases in diploid versus giant trophoblast cells. Interestingly, while cyclin B1 transcripts were found in both diploid and giant cells, the protein was found exclusively in diploid cells. This could be explained by either inhibition of translation or by constitutive degradation of the protein. The latter was ruled out by examining blastocysts which had been cultured in the presence of the proteasome inhibitor N-acetyl-leu-leu-norleucinal followed by immunostaining for cyclin B1. In these experiments cyclin B1 protein accumulated in diploid but not in giant cells. Fusion of trophoblast giant cells with secondary oocytes, which are rich in maturation promoting factor (MPF) activity, revealed that an exogenous source of active MPF could cause chromosome condensation and nuclear envelope breakdown in endocycling cells; therefore endoreduplication via polyteny evidently requires the suppression of MPF activity. In addition, cyclin D1 transcripts were found only in giant cells and, interestingly, the beginning of its expression was evident prior to that of placental lactogen I, an early marker of trophoblast differentiation. The results suggest that supression of MPF activity, by inhibition of translation of cyclin B1, is a key mechanism for the establishment of the endocycle in the mouse trophoblast.

Animals↗
PubMed1998

Complete dissipation of coherent clonal growth occurs before gastrulation in mouse epiblast.

Observations on chimaeric mice argue that there must be considerable dispersal and intermingling of the clonal descendants of epiblast founder cells at an early stage in the development of the tissue. However, it has not been established when or how this occurs. Here we have used a genetic marker that enables donor cells to be visualized in situ to examine the early postimplantation distribution of clones obtained by transplanting epiblast founder cells into host blastocysts. We have also determined the spatial relationship between sister cells in non-chimaeric postimplantation epiblast by ionophoretic injection of a fluorescent macromolecule. Both experimental approaches support the conclusion that breakdown of coherent clonal growth accompanies epithelialization of the epiblast and is essentially complete by the onset of gastrulation. Furthermore, the clonal analysis shows that descendants of different epiblast founder cells continue to intermingle extensively well into organogenesis. We suggest that this sustained intermingling of cells in the epithelial epiblast, which does not occur in the adjacent visceral endoderm, depends on cells losing contact with the basal lamina when they divide. These findings have implications both for patterning of the early amniote embryo and for the growth of tall columnar epithelia in general.

Animals↗
PubMed1998

Vascularization in the murine allantois occurs by vasculogenesis without accompanying erythropoiesis.

The aim of this study was to determine whether the blood vessels of the murine allantois are formed by vasculogenesis or angiogenesis. Morphological analysis revealed that differentiation of allantoic mesoderm into an outer layer of mesothelium and an inner vascular network begins in the distal region of the allantois, which is most remote from other tissues, as early as the late neural plate stage (approximately 7.75 days postcoitum). Nascent blood vessels were not found in the base of the allantois until 4-somite pairs had formed in the fetus (approximately 8.25 days postcoitum), and vascular continuity with the yolk sac and fetus was not present until the 6-somite-pair stage (approximately 8.5 days postcoitum). Immunohistochemical analysis demonstrated that flk-1, a molecular marker of early endothelial cells, is expressed in significantly more distal than basal core cells in the early allantois and never in mesothelium. Furthermore, synchronous grafting of donor yolk sac containing blood islands into blood islands of headfold-stage host conceptuses provided no evidence that the yolk sac contributes endothelial cells to the allantois. Finally, when removed from conceptuses and cultured in isolation, neural plate and headfold-stage allantoises formed a conspicuous vascular network that was positive for Flk-1. Hence, the vasculature of the allantois is formed intrinsically by vasculogenesis rather than extrinsically via angiogenesis from the adjacent yolk sac or fetus. Whether allantoic vasculogenesis is associated with erythropoiesis was also investigated. Benzidine-staining in situ revealed that primitive erythroid cells were not identified in the allantois until 6-somite pairs when continuity between its vasculature and that of the yolk sac was first evident. Nevertheless, a small number of allantoises removed from conceptuses at a considerably earlier stage were found to contain erythroid precursor cells following culture in isolation. To determine whether such erythroid cells could be of allantoic origin, host allantoises were made chimeric with lacZ-expressing donor allantoises that were additionally labeled with [3H]methyl thymidine. Following culture and autoradiography, many lacZ-expressing benzidine-stained cells were observed in donor allantoises, but none contained silver grains above background. Moreover, no cells of donor allantoic origin were found in the fetus or yolk sac. Hence, vasculogenesis seems to be independent of erythropoiesis in the allantois and to involve a distal-to-proximal gradient in differentiation of allantoic mesoderm into the endothelial cell lineage. Furthermore, this gradient is established earlier than reported previously, being present at the neural plate stage.

Allantois↗
PubMed1997

The origin and efficient derivation of embryonic stem cells in the mouse.

By explanting tissues isolated microsurgically from implanting strain 129 mouse blastocysts individually on STO feeder cells we have established that embryonic stem (ES) cells originate from the epiblast (primitive ectoderm). Isolated early epiblasts yielded ES cell lines at a substantially higher frequency than intact blastocysts regardless of whether they were explanted whole or as strictly single-cell suspensions. When explanted from delayed-implanting 129 blastocysts, epiblasts gave lines consistently in 100% of cases. If primary embryonic fibroblasts rather than STO cells were used as feeders, germline-competent ES cell lines were obtained readily from epiblasts of delayed-implanting blastocysts of several hitherto refractory strains, particularly when recombinant leukemia inhibitory factor was included in the medium during the initial period of culture. Because lines were obtained from the nonpermissive CBA/Ca strain at a rate of up to 56%, this approach to the derivation of germline-competent ES cell lines may not only prove generic for the mouse but also worth pursuing in other species of mammal.

Animals↗
PubMed1997

Relapse revisited.

Rather little is known about the changes in orthodontic treatment results exceeding a decade after treatment. The purpose of this study was to quantify changes in tooth relationships in a series of cases (n = 36) at 6 years and again at 15 years after treatment. The rate of change decreased with time, supporting the contention that most "relapse" occurs soon after treatment; continued change generally cannot be distinguished from normal aging processes that occur, regardless of whether a person had been treated orthodontically. There were minor, but statistically significant, associations between increased incisor irregularity ("relapse") and parasagittal growth of the jaws. Greater irregularity occurred when mandibular growth exceeded that of the maxilla, decreasing overjet and crowding the lower incisors within the containing arch of the maxilla. Overall, relapse tended to be less in these cases treated by a single experienced specialist that in university-based samples treated by multiple, orthodontic residents.

Adolescent↗
PubMed1997

The early blastocyst is bilaterally symmetrical and its axis of symmetry is aligned with the animal-vegetal axis of the zygote in the mouse.

At least one polar body, almost invariably the second, persists intact to the early blastocyst stage in nearly two-thirds of mouse conceptuses of the PO strain. The distribution in early blastocysts of these surviving polar bodies was highly non-random. Most not only lay in the mid-region of the embryonic-abembryonic axis but, on discovering that early blastocysts are bilaterally rather than radially symmetrical about this axis, were found to align with the bilateral axis. Cell marking experiments failed to detect movement of polar bodies relative to the surface of the conceptus during either cleavage or blastulation. That the distribution of degenerating polar bodies and their presumed debris was similar to intact ones also argued against their motility, as did the finding that at all stages second polar bodies were attached to conceptuses by a thin, extensible, weakly elastic 'tether'. Although the transfer of small fluorochromes between them was rarely observed beyond second cleavage, the second polar body and conceptus could remain coupled ionically up to the blastocyst stage. It is concluded that the second polar body normally remains attached to the conceptus through persistence of the intercellular bridge formed during its abstriction, and therefore provides an enduring marker of the animal pole of the zygote. Hence, according to the distribution of polar bodies, the axis of bilateral symmetry of the early blastocysts is normally aligned with the animal-vegetal axis of the zygote and its embryonic-abembryonic axis is orthogonal to it. Such relationships suggest that, at least in undisturbed development, specification of the axes of the blastocyst depends on spatial patterning of the zygote.

Animals↗
PubMed1997

Reflections on the biology of embryonic stem (ES) cells.

Remarkably little is known about mammalian embryonic stem (ES) cells despite their very widespread use in studies on gene disruption and transgenesis. As yet, it is only in the mouse that lines of ES cells which retain the ability to form gametes following reintroduction into the early conceptus have been obtained. Even in this species, most strains have so far proved refractory to the derivation of such cell lines. Apart from persisting ignorance as to how the various procedures that have been claimed to improve success actually do so, even the tissue of origin of ES remains uncertain. Furthermore, it is doubtful whether retention of pluripotency or expression of so-called "stem cell" marker molecules provide an adequate basis for classifying cells as genuine ES cells. This is because epiblast cells, their presumed precursors, lose the capacity to colonize the preimplantation conceptus well before they become restricted in the types of cell they can form or cease to express such marker molecules. In addition, it has yet to be established whether heterogeneity of cells within individual ES cell lines arises entirely during culture or is at least partly attributable to lack of homogeneity among their precursors. Finally, it has yet to be explained why ES chimeras evidently differ from those obtained by combining cells from different conceptuses in showing greater variation between tissues in the level of chimerism.

Animals↗
PubMed1996

Constitutive expression of the machinery for programmed cell death.

In the presence of cycloheximide (CHX) to inhibit protein synthesis, a high concentration of staurosporine (STS) induces almost all cells in explant cultures of 8/8 types of newborn mouse organs and 3/3 types of adult mouse organs to die with the characteristic features of apoptosis. Eggs and blastomeres also die in this way when treated with STS and CHX, although they are less sensitive to this treatment than trophectoderm or inner cell mass cells whose sensitivity resembles that of other developing cells. Human red blood cells are exceptional in being completely resistant to treatment with STS and CHX. As (STS plus CHX)-induced cell deaths have been shown to display the characteristic features of programmed cell death (PCD), we conclude that all mammalian nucleated cells are capable of undergoing PCD and constitutively express all the proteins required to do so. It seems that the machinery for PCD is in place and ready to run, even though its activation often depends on new RNA and protein synthesis.

Alkaloids↗
PubMed1996

Clonal analysis of growth of the polar trophectoderm in the mouse.

The results of experiments in which horseradish peroxidase (HRP) was used to mark single trophectoderm or inner cell mass (ICM) cells in situ in mouse blastocysts have led to the proposal that growth of the trophectoderm depends on stem cells located in the inner cell mass. Thus, the finding that the visual centre of clones formed following labelling of the central polar trophectoderm cell in early or expanding blastocysts was consistently shifted towards or into the mural trophectoderm was attributed to their displacement by ICM-derived cells. However, the frequency with which central polar cells were displaced is likely to have been overestimated by using the visual centre of descendant clones as the index of their location. Also, the possibility that displacement of central polar cells was an artefact of the marked temporary interruption of their cycling that resulted from labelling was not discounted. Furthermore, no attempt was made to ascertain whether cells located elsewhere in the polar trophectoderm also moved murally, as expected if there is a general displacement of such cells. In the present study, labelling of either the central or a peripheral polar trophectoderm cell with HRP was achieved without obviously perturbing their subsequent proliferation. Moreover, displacement was assessed by recording the location of the proximal boundary rather than the visual centre of the resulting clones. Even by this conservative criterion, the majority of labelled central cells moved towards or into the mural trophectoderm. In marked contrast, however, labelled peripheral polar cells moved murally in only a minority of cases. The remainder either retained their original position or moved towards rather than away from the central polar region. Such an anisotropic pattern of growth of the polar trophectoderm is not readily explicable in terms of recruitment of cells from the ICM. Rather, it accords with the view that the polar trophectoderm is a proliferative centre, and suggests that movement murally of its surplus cells may be restricted circumferentially, possibly through anchorage of the junctional trophectoderm cells that extend processes over the free surface of the ICM.

Animals↗
PubMed1995

Homozygosity for the Min allele of Apc results in disruption of mouse development prior to gastrulation.

Mutation of the APC (adenomatous polyposis coli) gene is an early event in colon tumor development in humans. Mice carrying Min (multiple intestinal neoplasia), a mutant allele of Apc, develop intestinal and mammary tumors as adults. To study the role of the Apc gene in development, we have investigated the phenotype of embryos homozygous for ApcMin (Min). Development of the primitive ectoderm fails prior to gastrulation in homozygous Min embryos. By midgestation, the presumed homozygotes consist of a mass of trophoblast giant cells with an additional cluster of much smaller embryonic cells. These results indicate that functional Apc is required for normal growth of inner cell mass derivatives.

Alleles↗
PubMed1995

Morphological changes in uterine leiomyomas treated by GnRH agonist goserelin.

To elucidate the mechanisms of action of GnRH agonists on uterine leiomyomas, morphometric and ultrastructural studies have been carried out on leiomyomas from patients treated with goserelin, and comparisons made with untreated tumors. The treated cases showed a variable degree of reduction of uterine size associated with leiomyoma shrinkage, and there was a correlation between the volume loss and the original size of the uterus. The morphological features of the treated lesions were very variable, with some showing evidence of marked cellular shrinkage and ultrastructural cytoplasmic changes, some having a lymphoid cell infiltrate, and others being indistinguishable from untreated control cases apart from having an increased number of compound lysosomes in the myoma cell cytoplasm. The degree of cellular shrinkage, measured by nuclear crowding, showed an inverse relationship to the proportion of collagen in the sections but none of the histological variables measured correlated with the degree of uterine shrinkage. It is concluded that the proportion of collagen in the lesions and other factors such as density of estrogen receptors, vascularity, cell proliferation rates, and degree of inflammatory cell infiltration affect the response to GnRH agonist therapy.

Adult↗
PubMed1995

An investigation into early placental ontogeny: allantoic attachment to the chorion is selective and developmentally regulated.

Culture of postimplantation conceptuses was used in conjunction with microsurgery to investigate the timing, the mechanism and the developmental regulation of chorioallantoic fusion in the mouse. The timing of fusion was determined in both freshly recovered conceptuses and in those that had been cultured from as early as the mid-streak stage. Attachment of the allantois to the chorion was found to have occurred in most conceptuses by the 6-somite stage, irrespective of whether they had been cultured. In investigating the mechanism of fusion, we wished to determine whether it depended on directed growth of the allantoic bud or on its differential adhesion to the chorion. Microsurgery was used to transplant allantoic tissue into the exocoelomic cavity of conceptuses from which the resident allantois had been removed. In synchronous grafting experiments, transplanted allantoises typically attached to the chorion despite loss of their connection with the hindgut region of the fetus. Hence selective attachment of the allantois to the chorion clearly cannot depend simply on its directed growth. While the transplanted allantoic tissue attached to the chorion selectively, it did not attach to it precociously, despite being favourably positioned to do so. These findings argue that the initial attachment of the allantois to the chorion depends on a selective adhesive mechanism that is developmentally regulated. Further grafting experiments in which donor conceptuses were either more or less advanced than hosts revealed that attachment of the allantois to the chorion depends primarily on the stage of the allantois rather than on the stage of the chorion. Collectively, these findings support the hypothesis that the initial stage of chorioallantoic fusion depends on selective adhesion between regionally differentiated mesodermal surfaces which is governed principally by the stage of development of the allantois.

Allantois↗