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R L Gardner

Publications and source records attributed to R L Gardner.

At least 19 recordsLinked to original sources
PubMed2006

The axis of polarity of the mouse blastocyst is specified before blastulation and independently of the zona pellucida.

BACKGROUND: Rather than being prepatterned, orientation of the embryonic-abembryonic (Em-Ab) axis of the mouse blastocyst has been claimed to depend on the conceptus being constrained by its zona pellucida (ZP) during blastulation. This hypothesis merited closer scrutiny, because it seemed at variance with observations on living conceptuses. METHODS: Two-cell conceptuses with an oil drop injected into the lesser diameter (LD) of the ZP at the first cleavage plane were cultured until shortly before blastulation when the blastomere underlying the drop was labelled with carbocyanine dye. After removing the ZP, conceptuses were re-cultured to the blastocyst stage for recording the position along the axis of the centres of the patches of labelled cells. RESULTS: These centres showed significant bias towards the equatorial (Eq) region of the axis compared with those resulting from labelling a blastomere at random, even following softening of the ZP at the 2-cell stage. This was also true if conceptuses were denuded at the 2-cell stage and the blastomere underlying an intact second polar body (PB) labelled in morulae. CONCLUSIONS: These findings further support the view that the Em-Ab axis of the mouse blastocyst is normally prepatterned and provide no evidence of a role for the ZP in its specification.

Animals↗
PubMed2006

Weaknesses in the case against prepatterning in the mouse.

Serious weaknesses are exposed regarding recent claims to have demonstrated that there is no prepatterning of axes in the mouse. That the orientation of the axis of polarity of the blastocyst is dictated by the shape of the zona pellucida is contradicted by systematic observations on living conceptuses. Moreover, this 'mechanical constraint' hypothesis fails to account for key findings on which the case for prepatterning is based.

Animals↗
PubMed2005

An investigation of the origin and significance of bilateral symmetry of the pronuclear zygote in the mouse.

BACKGROUND: Preliminary observations revealed that advanced zygotes of the PO strain mouse are often bilaterally symmetrical, and suggested that both the plane of first cleavage and features of the blastocyst bear a consistent relationship to the zygote's bilateral plane. METHODS: Spaced oil drops were injected into the zona pellucida to delineate the bilateral plane in pronuclear zygotes, and a distinct cluster of drops then placed over the second polar body. Such non-invasive marking was combined with gelation of the perivitelline space to prevent rotation of the zygotes within the zona pellucida. RESULTS: Nearly two-thirds of advanced pronuclear stage zygotes were bilaterally symmetrical and, regardless of whether first cleavage was meridional, it was almost invariably orthogonal to the bilateral plane. Moreover, both the axis of polarity and bilateral plane of the blastocyst bore a consistent relationship to the zygote's bilateral plane. Haploid parthenotes also exhibited bilateral symmetry, although in the absence of fertilization, first cleavage was less consistently orthogonal to the bilateral plane. CONCLUSIONS: Bilateral symmetry may be an intrinsic property of the oocyte that is induced by its activation and, from the reproducible way it maps on both the 2-cell conceptus and blastocyst, seems to play a role in early patterning.

Animals↗
PubMed2002

Patterning is initiated before cleavage in the mouse.

Once experimental embryological studies revealed the striking ability of mammals to regulate their early development, the notion that pattern-formation might depend on information already present in the egg before cleavage was generally regarded as untenable. Mammals were therefore assumed to differ from almost all other animals in the way in which their embryonic patterning was set up. This view was justified by the profound way in which their early development is modified to meet the requirements of viviparity. However, it ignored various findings showing that exposure of gametes and very early conceptuses to altered conditions could perturb organisation of the fetus. Recent studies that place particular emphasis on non-invasive approaches have revealed hitherto overlooked regularities in early mouse development. They clearly show that specification of embryonic axes normally begins before cleavage in this species. Moreover, the relevant patterning processes seem to depend on intrinsic organisation of the egg rather than, as claimed recently, the site of entry of the fertilizing sperm. These new findings are of interest for two reasons. First, from an evolutionary perspective, it means that mammals retain common features with other animals in how their early development is controlled. Second, it raises the practical question whether the increasing use of in vitro manipulation of gametes and zygotes for assisting human reproduction carries a risk of perturbing development.

Animals↗
PubMed2002

Thoughts and observations on patterning in early mammalian development.

The preimplantation mammalian conceptus shows an impressive ability to develop normally following the loss, gain or rearrangement of cells. This has prompted the view that, unlike in other species, patterning in mammals cannot depend on information that is already present in the zygote before it begins to cleave. However, various findings are hard to reconcile with this conclusion, including evidence that the incidence of monozygotic twinning is sensitive to the conditions to which eggs or very early concepti are exposed. Possible causes of early twinning are discussed, and it is argued that partial hatching of the conceptus through a hernia in the zona pellucida cannot account for all cases. Moreover, it remains questionable whether studies on aggregated morulae and isolated blastomeres really provide compelling evidence against the existence of indispensible patterning information in the egg. Finally, regularities in axial relationships between the blastocyst and zygote have been revealed employing strictly non-invasive techniques. These show that, at least in normal development, patterning begins before cleavage.

Animals↗
PubMed2002

Stem cells: potency, plasticity and public perception.

Production of chimaeras with embryonal carcinoma and embryonic stem cells enabled a very thorough investigation of the potency of these cells in the mouse. Human embryonal carcinoma and embryonic stem cell differ from their murine counterparts in a number of respects and, for obvious reasons, their potency is more difficult to assess. Recently, findings attesting to a surprising degree of plasticity of cells from adults have begun to emerge, which, aside from offering a possible further route to stem cell therapy, raise intriguing questions about the importance of lineage in the process of cellular diversification. Biomedical research is widely perceived to be advancing too fast to allow proper consideration of the implications of its clinical applications. Whilst this was clearly not true in the case of human in vitro fertilization, it has some validity regarding stem cell therapy, even though many of the issues are common to both. Casual use of the term 'embryo' proved unhelpful in the past debate on whether research on early stages of human development should be permitted. Likewise, introduction of the term 'therapeutic cloning' has complicated the present one regarding extension of such research to stem cell therapy.

Adult↗
PubMed2002

Experimental analysis of second cleavage in the mouse.

BACKGROUND: Mammalian conceptuses typically have an approximately regular tetrahedral shape at the 4-cell stage. In the rabbit, this has been attributed to both 2-cell blastomeres dividing meridionally, but with the animal-vegetal axis of the second blastomere to divide rotating through roughly 90 degrees before or during cytokinesis. The aim of the present study was to ascertain whether this was also true for the mouse. METHODS AND RESULTS: First, the distribution in regular tetrahedral 4-cell conceptuses of fluorescent microspheres applied to the vegetal polar region of one or both blastomeres at the 2-cell stage was analysed. Second, the ability of 2-cell stages to form regular tetrahedral 4-cell conceptuses after the previtelline space had been gelated to prevent blastomeres from rotating was also investigated. Neither experiment yielded evidence supporting blastomere rotation during second cleavage. Rather, the findings were consistent with the regular tetrahedral form of 4-cell conceptus resulting from meridional division of one blastomere and approximately equatorial division of the other. CONCLUSIONS: Second cleavage in the mouse typically yields 4-cell conceptuses with three distinct types of blastomere. While both products of the meridional division include all axial levels of the zygote, those of the equatorial division acquire only its vegetal or animal half.

Animals↗
PubMed2002

The plane of first cleavage is not related to the distribution of sperm components in the mouse.

BACKGROUND: Marking experiments using phytohaemagglutinin (PHA) and Concanavalin A (ConA) have suggested that the first cleavage plane is related to the point of sperm entry. Because of concerns about the specificity of lectin binding, the distribution of sperm components has been investigated directly. METHODS AND RESULTS: The sperm tail could be identified in cleaving zygotes and early 2-cell stages following their permeabilization and exposure to Oregon Green Paclitaxel. At neither stage did the anterior end of the tail, which lies initially at the site of sperm entry, bear a consistent relationship to the first cleavage plane, even when it had clearly retained its original location. Moreover, using artificial insemination with MitoTracker-labelled sperm, the midpiece was found to remain associated with anterior end of the tail through to the 2-cell stage. Lectins showed no discernible binding to the fertilization cone of mechanically denuded zygotes and very strong binding to the zona pellucida. Moreover, after general labelling of zygotes with either ConA or PHA, persisting surface lectin tended to be concentrated towards the cleavage plane. CONCLUSION: The present findings challenge the claim that the sperm specifies the plane of first cleavage, and also question the methodology on which it was based.

Animals↗
PubMed2001

The initial phase of embryonic patterning in mammals.

Although specification of the antero-posterior axis is a critical intial step in development of the fetus, it is not known either how, or at what stage in development, this process begins. Such information is vital for understanding not only normal development in mammals but also monozygotic twinning, which, at least in man, is associated with a significantly increased incidence of birth defects. According to recent studies in the mouse, specification of the fetal anteroposterior axis begins well before gastrulation, and probably even before the conceptus implants. Moreover, evidence is accruing that the origin of relevant asymmetries depends on information that is already present in the zygote before it embarks on cleavage. Hence, early development in mammals does not differ as markedly from that in other animals as has generally been assumed. Consequently, at present, the possibility of adverse effects of techniques used to assist human reproduction cannot be disregarded.

Animals↗
PubMed2001

Specification of embryonic axes begins before cleavage in normal mouse development.

Studies on the development of aggregated, isolated and rearranged blastomeres have engendered the view that in mammals, unlike most other animals, egg organization has no role in the genesis of asymmetries that are essential for cellular diversification and the specification of embryonic axes. Such asymmetries are assumed to arise post-zygotically through interactions between initially naive cells. However, various findings are difficult to reconcile with this view. Here, a consistent relationship between the structure of the blastocyst and the two-cell stage in the mouse has been found using a strictly non-invasive marking technique: injection of small oil drops into the substance of the zona pellicuda. This has revealed that both the embryonic-abembryonic axis of the blastocyst and its plane of bilateral symmetry are normally orthogonal to the plane of first cleavage. This relationship was also seen when denuded two-cell conceptuses were prevented from rotating during subsequent cleavage by immobilizing them in a gel. Therefore, during normal mouse development the axes of the blastocyst, which have been implicated in establishing those of the fetus, are already specified by the onset of cleavage.

Animals↗
PubMed2000

Directed differentiation of dendritic cells from mouse embryonic stem cells.

Dendritic cells (DCs) are uniquely capable of presenting antigen to naive T cells, either eliciting immunity [1] or ensuring self-tolerance [2]. This property identifies DCs as potential candidates for enhancing responses to foreign [3] and tumour antigens [4], and as targets for immune intervention in the treatment of autoimmunity and allograft rejection [1]. Realisation of their therapeutic potential would be greatly facilitated by a fuller understanding of the function of DC-specific genes, a goal that has frequently proven elusive because of the paucity of stable lines of DCs that retain their unique properties, and the inherent resistance of primary DCs to genetic modification. Protocols for the genetic manipulation of embryonic stem (ES) cells are, by contrast, well established [5], as is their capacity to differentiate into a wide variety of cell types in vitro, including many of hematopoietic origin [6]. Here, we report the establishment, from mouse ES cells, of long-term cultures of immature DCs that share many characteristics with macrophages, but acquire, upon maturation, the allostimulatory capacity and surface phenotype of classical DCs, including expression of CD11c, major histocompatibility complex (MHC) class II and co-stimulatory molecules. This novel source should prove valuable for the generation of primary, untransformed DCs in which candidate genes have been overexpressed or functionally ablated, while providing insights into the earliest stages of DC ontogeny.

Animals↗
PubMed2000

A structurally defined mini-chromosome vector for the mouse germ line.

Yeast artificial mini-chromosomes have helped to define the features of chromosome architecture important for accurate segregation and replication and have been used to identify genes important for chromosome stability and as large-fragment cloning vectors. Artificial chromosomes have been developed in human cells but they do not have defined, experimentally predictable structures. Fragments of human chromosomes have also been introduced into mice and in one case passed through the germ line. In these experiments, however, the structure and sequence organization of the fragments was not defined. Structurally defined mammalian mini-chromosome vectors should allow large tracts of DNA to be introduced into the vertebrate germ line for biotechnological purposes and for investigations of features of chromosome structure that influence gene expression. Here, we have determined the structure and sequence organization of an engineered mammalian mini-chromosome, ST1, and shown that it is stably maintained in vertebrate somatic cells and that it can be transmitted through the mouse germ line.

Animals↗
PubMed2000

Flow of cells from polar to mural trophectoderm is polarized in the mouse blastocyst.

During growth of the blastocyst there is a net flow of cells from the polar to the mural trophectoderm which is presumed to be radially symmetrical. However, such a pattern of cell movement is inconsistent with findings from a recent clonal analysis. To visualize the overall flow of cells directly, the polar trophectoderm of expanding blastocysts was labelled globally with fluorescent microspheres. Following further growth, the great majority of blastocysts that remained labelled throughout the polar trophectoderm exhibited a polarized rather than radial spread of label into the mural region. This was the case regardless of the labelling technique, whether the blastocysts were grown in utero or in vitro, or had the zona pellucida removed or left on. Intriguingly, where there were two foci of spread of label into the mural trophectoderm rather than one, these were diametrically opposite each other. In further experiments, fluorescent lineage labels were used to distinguish junctional trophectoderm cells with and without an extension onto the blastocoelic surface of the inner cell mass. The location of clones formed following further blastocyst growth provided no evidence that egress of cells from the polar trophectoderm is restricted circumferentially by the presence of junctional cells having an extension.

Animals↗
PubMed1999

Synthesis and NMR assignments of galactosylgloboside and its beta-D-GalNAc-(1-->4)-alpha-D-Gal-linked positional isomer in a conjugatable form.

Two pentasaccharides suitable for conjugation, namely 3-aminopropyl glactosylgloboside and its beta-D-GalNAc-(1-->4)-alpha-D-Gal-linked positional isomer, were synthesized from 3III,4III-di-O-unprotected globotrioside and the trichloroacetimidate of beta-D-Gal-(1-->3)-beta-D-GalNPhth derivative. Glycosylation at both positions led to the formation of beta-D-GalNPhth-(1-->4)-alpha-D-Gal and beta-D-GalNPhth-(1-->3)-alpha-D-Gal-linked products in a ratio of 1:1 without selectivity. Complete NMR spectral assignments are also described.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed1999

Scrambled or bisected mouse eggs and the basis of patterning in mammals.

Several findings challenge the notion that specification of cell types and embryonic axes in mammals are rooted entirely in the temporal and spatial relations between cleaving blastomeres. They raise the question as to whether, as in most non-mammalian species, these processes depend on information already present in the egg. However, experiments designed to investigate this possibility directly by perturbing the organization of the zygote or, very recently, by deleting one or other of its polar regions [M. Zernicka-Goetz. Fertile offspring derived from mammalian eggs lacking either animal or vegetal poles. Development 1998;125:4803-4808 (Ref. 1)], have been interpreted to mean that such a role for the egg can be discounted. This conclusion seems premature in view of continuing uncertainty regarding the developmental potential of individual blastomeres in mammals.

Animals↗