Prenatal fate of parthenogenetic cells in mouse aggregation chimaeras

Parthenogenetically activated BCF1 and fertilized BALB/c embryos were aggregated to form chimaeras. The fate of the parthenogenetic component was followed in the conceptus during the second half of gestation. The results indicate an early strong selection against parthenogenetic cells in the extra-embryonal part, which is presumably complete by term, and a weaker selective process in the embryo. During early development, parthenogenetic cells have nearly normal developmental potency in the embryo, which allows their balanced contribution in the chimaeras on day 12. Later, this contribution declines significantly resulting in an unbalanced relation to the advantage of the fertilized counterpart. From the results, we suggest that gametic imprinting may play a role not only in the key steps of preimplantation and early postimplantation development, but later in cell and tissue differentiation.     

Systematic elimination of parthenogenetic cells in mouse chimeras

The developmental potential of primitive ectoderm cells lacking paternal chromosomes was investigated by examining the distribution of parthenogenetic cells in chimeras. Using GPI-1 allozymes as marker, parthenogenetic cells were detected in most organs and tissues in adult chimeras. However, these cells were under severe selective pressure compared with cells from normal fertilized embryos. In the majority of chimeras, parthenogenetic cells in individual animals were observed in a limited number of tissues and organs and, even in these instances, their contribution was substantially reduced. Nevertheless, parthenogenetic cells were detected more consistently in some organs, especially the brain, heart, kidney and spleen. In contrast, there was apparently a systematic selection against parthenogenetic cells in some tissues, most notably in skeletal muscle, liver and pancreas. These results suggest that paternally derived genes are probably required not only for the development of extraembryonic structures but also for subsequent development of embryonic tissues derived from the primitive ectoderm lineage.     

XY follicle cells in ovaries of XX----XY female mouse chimaeras

Oocytes with adhering follicle cells were sampled from ovaries obtained from 11 GPI-1A----GPI-1B chimaeras, comprising 10 females and 1 hermaphrodite. GPI analysis of individual oocytes revealed a marked bias towards the GPI-1B component in the germ line of this chimaeric combination. GPI-1B XY oocytes were identified in the ovary from the hermaphrodite, the bias towards the GPI-1B germ line perhaps helping to counterbalance the normally severe selection against XY oocytes. GPI analysis of follicle cells revealed a much more balanced contribution of the two components to this ovarian cell type. Importantly, GPI-1A follicle cells were identified in more than half the follicles from an XX----XY female in which the GPI-1A component was XY, supporting an earlier conclusion of Ford et al. (1974) that XY cells can contribute to the follicles of XX----XY female mice. It is suggested that XY cells can be recruited to form follicle cells in XX----XY chimaeras when there is a developmental mismatch between the two components, such that an ovary-determining signal produced by the XX component pre-empts the testis-determining action of the Y.     

Distinctive left-sided distribution of adrenergic-derived cells in the adult mouse heart

Adrenaline and noradrenaline are produced within the heart from neuronal and non-neuronal sources. These adrenergic hormones have profound effects on cardiovascular development and function, yet relatively little information is available about the specific tissue distribution of adrenergic cells within the adult heart. The purpose of the present study was to define the anatomical localization of cells derived from an adrenergic lineage within the adult heart. To accomplish this, we performed genetic fate-mapping experiments where mice with the cre-recombinase (Cre) gene inserted into the phenylethanolamine-n-methyltransferase (Pnmt) locus were cross-mated with homozygous Rosa26 reporter (R26R) mice. Because Pnmt serves as a marker gene for adrenergic cells, offspring from these matings express the β-galactosidase (βGAL) reporter gene in cells of an adrenergic lineage. βGAL expression was found throughout the adult mouse heart, but was predominantly (89%) located in the left atrium (LA) and ventricle (LV) (p<0.001 compared to RA and RV), where many of these cells appeared to have cardiomyocyte-like morphological and structural characteristics. The staining pattern in the LA was diffuse, but the LV free wall displayed intermittent non-random staining that extended from the apex to the base of the heart, including heavy staining of the anterior papillary muscle along its perimeter. Three-dimensional computer-aided reconstruction of XGAL+ staining revealed distribution throughout the LA and LV, with specific finger-like projections apparent near the mid and apical regions of the LV free wall. These data indicate that adrenergic-derived cells display distinctive left-sided distribution patterns in the adult mouse heart.     

Fate of haploid parthenogenetic cells in mouse chimeras during development

The developmental capability of haploid parthenogenetic cells was investigated by studies on haploid parthenogenetic in equilibrium fertilized mouse chimeras. Two chimeras were born. One female chimera was smaller at birth and grew slower than its littermates. The distribution of haploid-derived cells in the chimeras was analyzed 11 months after their birth. Cells derived from haploid embryos were found only in the brain, eyes, pigment cells in hair follicles, and spleen, in which they constituted 30%, 20%, 10%, and less than 5%, respectively, of the cells. The correlation between the parthenogenetic contribution to the brain and growth retardation is discussed. All of the cells examined in these chimeric organs (brain and eyes) contained a diploid amount of DNA, suggesting that diploidization of the haploid parthenogenetic cells occurred during development. Possibly, the haploid state is not sufficient for cell growth, even in chimeras with fertilized embryos.     

Germline potential of parthenogenetic haploid mouse embryonic stem cells

Haploid embryonic stem cells (ESCs) have recently been derived from parthenogenetic mouse embryos and offer new possibilities for genetic screens. The ability of haploid ESCs to give rise to a wide range of differentiated cell types in the embryo and in vitro has been demonstrated. However, it has remained unclear whether haploid ESCs can contribute to the germline. Here, we show that parthenogenetic haploid ESCs at high passage have robust germline competence enabling the production of transgenic mouse strains from genetically modified haploid ESCs. We also show that differentiation of haploid ESCs in the embryo correlates with the gain of a diploid karyotype and that diploidisation is the result of endoreduplication and not cell fusion. By contrast, we find that a haploid karyotype is maintained when differentiation to an extra-embryonic fate is forced by induction of Gata6.     

Differentiation diversity of mouse parthenogenetic embryonic stem cells in chimeric mice

Recent studies have illustrated multiple differentiation potentials of embryonic stem cells (ESCs), derived from parthenogenetic embryos, to various kinds of cells (all three embryonic germ layers). However, differentiation diversity of the parthenogenetic ESCs (PgESCs) in vivo remains to be elucidated. In the present study, we established mouse PgESC-lines and observed their contribution diversity in vivo by producing chimeric mice using embryos possessing single nucleotide polymorphisms of mitochondrial DNA (mtDNA) as hosts. Based on southern blot analysis using specific probes to detect the SNPs on mtDNA, PgESC-derived mtDNA were contained in many organs such as brain, lung, and heart of the chimeric mouse. We concluded that PgESCs contributed to various internal organs in vivo, and that they were also stably maintained in adult animals.     

Developmental incompatibility of human parthenogenetic embryonic stem cells in mouse blastocysts

Human parthenogenetic embryonic stem (pES) cells can be clinically used in the future to avoid immunological rejection. However, the developmental potential of human pES cells remains to be elucidated. In this study, we generated a human pES-enhanced green fluorescent protein (EGFP) cell line (chHES-32-EGFP), which shows pluripotency thus far and maintains stable and robust EGFP expression in the undifferentiated and differentiated states in vivo and in vitro. Using this pES-EGFP cell line, we found that when human pES-EGFP cells were injected into mice blastocysts, EGFP-positive cells progressively decreased with the development of blastocysts in vitro. Only 4 out of 23 embryos (17.4%) contained EGFP-positive cells and all of these embryos exhibited abnormal morphology or delayed development when the chimera blastocysts were implanted into the pseudopregnant recipient mouse uterus. These results raise serious questions regarding the feasibility of the generation of interspecific chimeras between mouse blastocysts and human pES cells.     

Immunohistochemistry in the analysis of mouse aggregation chimaeras

Summary We have used cellular mosaicism in chimaeric mice to study the clonal organization of normal tissues. The mosaicism has been demonstrated in sections and in whole mounts of intestinal epithelium, aortic endothelium and retinal pigment epithelium using H2 antigens and a carbohydrate polymorphism recognized byDolichos biflorus lectin as strain-specific markers.The results show that the epithelium of each adult intestinal crypt is derived from a single progenitor cell. Because crypts of differing genotype may contribute cells to the same villus, the pathways of cell migration up the villi can be demonstrated. The ability to stain mosaic patches in two dimensions in large intact sheets of epithelium has permitted a more satisfactory analysis in terms of clonal development than was previously possible with data from tissue sections. We have adapted statistical procedures from plant ecology to examine the scale of clustering of patches of like genotype, and thence to recognize descendent clones, i.e. groups of cells which are not contiguous, but are related by descent from a common ancestor in embryogenesis.The 1985Histochemical Journal Lecture given by Dr Ponder at York on 10 July, 1985 at the invitation of the Royal Microscopical Society.     

Tolerance in early embryo aggregation-derived mouse chimaeras.

Epidermal cell suspensions of 90% average viability prepared from adult mouse tail skin by trypsinization and glass wool filtration were compared with leucocytes for their capacity to induce and reveal cell-mediated cytotoxicity to histocompatibility antigens in an H-2 different strain combination. As targets in short-term chromium release assays, epidermal cells incorporated ten times more ⁵¹Cr than normal lymph node cells yet released a lower proportion spontaneously. Although they were more resistant to lysis than lymph node cells, they registered high levels of cytotoxicity when particularly active attacker cells were used, even at low attacker-to-target cell ratios. In mixed cell cultures, irradiated epidermal cells were as effective as spleen cells in boosting immunity induced in vivo by skin allografts. Epidermal cells also were effective primary immunogens in vitro, but at higher responder-to-stimulator cell ratios than required for spleen cells. The specificity of epidermal cells as both targets and immunogens fully paralleled that of leucocytes from the same donors.     

The histochemical identification of primordial germ cells in diploid parthenogenetic mouse embryos

An attempt has been made to improve the early post-implantation development potential of diploid parthenogenetic mouse embryos by transferring parthenogenetic blastocysts to one uterine horn of a pseudopregnant recipient and a similar number of fertilized embryos to the contralateral horn. In control studies, diploid parthenogenetic embryos were transferred to both uterine horns of appropriate recipients. Unfortunately no obvious advantage appeared to be gained by carrying out the former manoeuvre. A significant improvement in the development potential of the parthenogenones could have indicated that their poor post-implantation survival might have been associated with a deficiency, possibly of hormonal origin, in the functioning of their decidual reaction. However, sufficient somite-containing parthenogenetic embryos were obtained in this study to allow a comparison to be made between them and fertilized embryos that were morphologically at a comparable stage of development. The parthenogenones were found to have a markedly smaller crown-rump length than their fertilized counterparts. A high proportion of both the parthenogenetic and fertilized embryos were subsequently fixed and appropriately stained in order to localize alkaline phosphatase activity. The analysis of this material clearly demonstrated that parthenogenetic mouse embryos are in fact capable of producing primordial germ cells. The latter were recognized by their morphology, histochemical staining appearance, and characteristic location, being found in the early 'turned' embryos within the dorsal mesentery in close proximity to the developing gut tube, and in the more advanced limb-bud stage embryos within the gonadal ridges.     

Tissue specific loss of proliferative capacity of parthenogenetic cells in fetal mouse chimeras

Parthenogenetic cells are lost from fetal chimeras. This may be due to decreased proliferative potential. To address this question, we have made use of combined cell lineage and cell proliferation analysis. Thus, the incorporation of bromodeoxyuridine in S-phase was determined for both parthenogenetic and normal cells in several tissues of fetal day 13 and 17 chimeras. A pronounced reduction of bromodesoxyuridine incorporation by parthenogenetic cells at both developmental stages was only observed in cartilage. In brain, skeletal muscle, heart and intestinal epithelium, this reduction was either less pronounced or observed only at one of the developmental stages analysed. No difference between parthenogenetic and normal cells was observed in epidermis and ganglia. Our results show that a loss of proliferative potential of parthenogenetic cells during fetal development contributes to their rapid elimination in some tissues. The analysis of the fate of parthenogenetic cells in skeletal muscle and cartilage development demonstrated different selection mechanisms in these tissues. In skeletal muscle, parthenogenetic cells were largely excluded from the myogenic lineage proper by early post-midgestation. In primary hyaline cartilage, parthenogenetic cells persisted into adulthood but were lost from cartilages that undergo ossification during late fetal development.     

Polyclonal origin of pancreatic islets in aggregation mouse chimaeras.

In the present study, we have examined the origin and growth pattern of the beta cells in pancreatic islets, to determine whether a single progenitor cell gave rise to all the precursors of the islets, or if each of a few progenitor cells is the founder of a different islet, or if each islet is a mixture of cells originating from a pool of progenitor cells. Aggregation mouse chimaeras where the pancreatic beta cells derived from each embryo can be identified in the islets on histological sections were analyzed. In two chimaeras, all the islets contained cells from both the aggregated embryo. This clearly demonstrates that each islet resulted from several independent cells. In addition, the beta cells derived from either embryo component were in very small clusters in the islets, suggesting that in situ cell division did not account significantly for islet growth.     

Studies of Sl/Sld in equilibrium with +/+ mouse aggregation chimaeras. I. Different distribution patterns between melanocytes and mast cells in the skin

In spite of their different origin, both melanocytes and mast cells are deficient in the skin of mutant mice of the Sl/Sld genotype. Since the neural crest and the liver of Sl/Sld embryos contain normal precursors of melanocytes and mast cells, respectively, the deficiency is attributed to a defect in tissue environment necessary for migration and/or differentiation of precursor cells. We investigated whether the tissue environment used for differentiation of melanocytes and mast cells was identical by producing aggregation chimaeras from Sl/Sld and +/+ embryos. Chimaeric mice with apparent pigmented and nonpigmented stripes were obtained. In the nonpigmented stripes of these Sl/Sld in equilibrium with +/+ chimaeras, melanocytes were not detectable in hair follicles but were detectable in the dermis. In contrast, melanocytes were detectable neither in hair follicles nor in the dermis of nonchimaeric Sl/Sld mice. Concentrations of mast cells were comparable in the pigmented and nonpigmented stripes of Sl/Sld in equilibrium with +/+ chimaeras, but the average concentration of mast cells significantly varied in the chimaeras (from 8% to 74% of the value observed in control +/+ mice). The present result suggests that mesodermal cells that support the migration and differentiation of both melanocyte precursors and mast-cell precursors mix homogeneously in the dermis and that ectodermal cells that influence the invasion of differentiating melanocytes into hair follicles make discrete patches.     

Subnormal zona pellucida change in parthenogenetic mouse embryos

Parthenogenetic mouse embryos were obtained by the method of electrical stimulation of eggs in vivo(Tarkowski et al., 1970), and their developmental retardation and limited viability were confirmed. Very early deviations from normalcy seemed likely in these embryos, and we chose to investigate their “zona reaction,” as this is one of the earliest events identified (Braden, et al., 1954) in normal fertilization. The change, ordinarily triggered by sperm penetration of the egg, decreases the permeability of the zona pellucida to supernumerary sperms, and has been attributed (Austin and Braden, 1956) to products released by cortical granules.An indirect assay for the state of the zona pellucida is presented. It is based on the observation (Mintz, 1970) that pronase, other proteolytic enzymes, and the normal uterine zonalytic factor lyse zonas of fertilized eggs more slowly than those of unfertilized eggs. Comparative zona lysis times in pronase are thus employed as a test for the degree of zona change after parthenogenetic activation relative to that after activation by sperm.The zonas of parthenogenetic embryos in stages ranging from 2 to 14 cells varied in their lysis times in pronase and overlapped with those of unfertilized and fertilized egg zonas. As a population, the zonas of the parthenogenones had intermediate lysis times. Thus, in the strains tested, electrical shock evokes only a partial zona reaction and, in this respect, is not an adequate substitute for sperm penetration.A working hypothesis for future testing is that the subnormal zona change and retarded development may both be due to inadequate release of products from cortical granules, under these conditions of artificial activation of the mouse egg.     

The nuclear-cytoplasmic relationship in 'mosaic' skeletal muscle fibers from mouse chimaeras

In mouse chimaeras, individual skeletal muscle fibers typically contain populations of myonuclei derived from both cell lines. This 'mosaic' circumstance has provided an opportunity to investigate directly whether the mammalian myofiber syncytium is functionally subdivided into territories, each preferentially influenced by products encoded by the local myonucleus, or whether the multiple nuclei direct the synthesis of products that achieve a uniform distribution throughout the fiber. Chimaeras were produced in which one cell line was derived from an embryo homozygous for gpi-1a, whereas the other was homozygous for the gpi-1b; each allele specifies electrophoretically distinguishable isozymes of the cytosolic enzyme glucosephosphate isomerase (GPI-1). Microtechniques capable of measuring the proportion of each isozyme expressed within small samples of individual muscle fibers have been established, permitting the comparison of the relative quantitative distributions of the GPI-1 isozyme types along the length of individual chimaera fibers. From individual mosaic fibers, all samples yielded identical isozyme profiles, demonstrating that GPI-1 is not sequestered adjacent to the nucleus directing its synthesis; rather, it achieves a homogeneous distribution throughout the mosaic syncytium. The GPI-1 gene locus encodes only the GPI-1 monomer, whereas the functional enzyme detected in our analysis is a dimer that results from the aggregation of monomers in the cytoplasm. The quantitative distribution of dimer types within each mosaic fiber was consistent with random aggregation amongst all monomers represented in the final isozyme pattern, a result requiring that monomers or earlier precursors were mixed in the myofiber cytoplasm prior to assembly of the enzymatically active dimer. Thus, both the final distribution of enzyme dimers within fibers and the patterns of monomer aggregation suggest that there are no subdivisions related to the spatial separation of the genotypically distinct myonuclei within mosaic muscle fibers.     

DNA synthesis and distribution in parthenogenetic bovine embryos

Parthenogenetically activated, in vitro-matured bovine oocytes and parthenogenotes obtained at 2 to 4 days post activation were analyzed by 3H-thymidine autoradiography for the timing of the S-phase and for distribution of newly replicated DNA, respectively. Spread pronuclear parthenogenotes revealed that the DNA synthesis in electrically stimulated oocytes commenced at 14 h post activation. At 20 to 24 h, a maximum number of labeled pronuclei was reached (25 to 38%), and DNA synthesis persisted in some parthenogenotes up to 30 h post activation. The DNA labeling detected on semi-thin sections showed that the distribution of newly synthesized DNA in the nuclei of 3- to 16-cell parthenogenotes was mostly irregular or abnormal, documenting that the apparent morphological normalcy of parthenogenotes was in contrast to the data concerning the DNA synthesis and distribution.     

Transient reactivation of CSF in parthenogenetic one-cell mouse embryos

During meiosis, the cytostatic factor (CSF) activity stabilizes the activity of the M-phase promoting factor (MPF) in metaphase II arrested vertebrate oocytes. Upon oocyte activation, the inactivation of both MPF and CSF enables the entry into the first embryonic mitotic cell cycle. Using a biological assay based on cell-fusion (hybrid between a parthenogenetically activated egg entering the first mitotic division and an activated oocyte), we observed that in activated mouse oocytes a first drop in CSF activity is detectable as early as 20 min post-activation. This suggests that CSF is inactivated upon MPF inactivation. However, CSF activity increases again to reach a maximum 60 min post-activation and gradually disappears during the following 40 min. Thus, in activated mouse oocytes (undergoing the transition to interphase) CSF activity fluctuates before definitive inactivation. We found that hybrids arrested in M-phase, thus containing CSF activity after oocyte activation, have activated forms of MAP kinases while hybrids in interphase have inactive forms of these enzymes. We postulate that CSF inactivation in mouse oocytes proceeds in two steps. The initial inactivation of CSF, required for MPF inactivation, is transient and does not require MAP kinase inactivation. The final inactivation of CSF, required for normal embryonic cell cycle progression, is dependent upon the inactivation of MAP kinases.