Temporal and spatial selection against parthenogenetic cells during development of fetal chimeras

The fate of parthenogenetic cells was investigated during development of fetal and early postnatal chimeras. On day 13 of embryonic development, considerable contribution of parthenogenetic cells was observed in all tissues of chimeric embryos, although selection against parthenogenetic cells seemed to start before day 13. Between days 13 and 15 of development, parthenogenetic cells came under severe selective pressure, which was most striking in tongue. The disappearance of parthenogenetic cells from tongue coincided with the beginning of myoblast fusion in this tissue. Severe selection against parthenogenetic cells was also observed in pancreas and liver, although in the latter, parthenogenetic cells were eliminated later than in skeletal muscle or pancreas. In other tissues, parthenogenetic cells may persist and participate to a considerable extent throughout the gestation period and beyond, although a significant decrease was observed in all tissues. Parthenogenetic in equilibrium fertilized chimeras were significantly smaller than their non-chimeric littermates at all developmental stages. These results suggest that the absence of paternal chromosomes is largely incompatible with the maintenance of specific differentiated cell types. Furthermore, paternally derived genes seem to be involved in the regulation of proliferation of all cell types, as indicated by the drastic growth decceleration of parthenogenetic in equilibrium fertilized chimeras and the overall decrease of parthenogenetic cells during fetal development. Chromosomal imprinting may have a role in maintaining a balance between cell growth and differentiation during embryonic development. The major exception to the selective elimination of parthenogenetic cells appear to be the germ cells; viable offspring derived from parthenogenetic oocytes were detected, sometimes at a high frequency in litters of female parthenogenetic in equilibrium fertilized chimeras.     

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.     

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.     

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.     

An analysis of parthogenetic cell clones in chimeric C57BL/6(PG)<-->BALB/c mice

We studied the distribution of parthenogenetic cell clones in the retinal pigment epithelium and choroid of eyes on serial sections and in the brain, kidneys, and liver by electrophoretic analysis of glucose phosphate isomerase isozymes in 12 mouse chimeras C57BL/6(PG)<-->BALB/c obtained earlier. Asymmetry was noted in the distribution of the parthenogenetic cell clones in the eye structure, just as the earlier established asymmetry in the distribution of the parthenogenetic clones of epidermal melanoblasts. A high correlation was shown between the ratio of parthenogenetic to normal cells in the retinal pigment epithelium of the right or left eyes and epidermal melanoblasts in the hair cover of the corresponding body half of the chimera. These data suggest that there is a certain relationship between the processes leading to the characteristic distribution of the ectodermal parthenogenetic clones in the retinal pigment epithelium of the right and left eyes and epidermal melanoblasts in parthenogenetic chimeras. Electrophoretic analysis did not show parthenogenetic components in the liver or kidneys of any chimera, and the parthenogenetic component was found in the brain of only two chimeras, in which a high percentage of parthenogenetic cells of ectodermal origin was noted. In these cases, asymmetry was noted in the right and left cerebral hemispheres, just as in the retinal pigment epithelium of the right and left eyes. The data obtained suggest that, during the development of the chimeras, parthenogenetic C57BL/6 cells were actively eliminated from the tissues of endodermal and mesodermal origin. In adult chimeras C57BL/6(PG)<-->BALB/c, parthenogenetic cell clones of ectodermal origin are mostly preserved.     

Teratogenic effects of parthenogenetic cells from LTXBO mice,a strain which develops ovarian teratomas at high frequency

Summary LTXBO mice develop ovarian teratomas at high frequency. The phenotype of tumour tissues is unusual in that most contain trophoblast elements. Since the tumours are derived from parthenogenetically activated oocytes, they would not be expected to produce trophoblast. The developmental potential of parthenogenetic cells from these mice was tested in aggregation chimeras. No contribution to trophoblast tissues was observed. However, a high incidence of morphological abnormalities was seen, suggesting that the parthenogenetic cells exerted a teratogenic effect.     

Developmental potential of parthenogenetic cells: role of genotype-specific modifiers.

The developmental potential of parthenogenetic cells derived from different mouse strains was investigated by examining their distribution in various tissues of adult aggregation chimeras. Using GPI-1 allozymes as marker, no striking differences were observed between chimeras whose parthenogenetic cells were derived from activated oocytes isolated from females of different genetic backgrounds, (C57BL/6 x CBA/J) F1, CFLP, 129, and SWR. In all the combinations tested, parthenogenetic cells were consistently absent from skeletal muscle, but there were varying contributions to most other tissues. These results suggest that the maternal duplication of chromosomes containing imprinted gene(s) responsible for the systematic elimination of parthenogenetic cells from skeletal muscle, are not subject to a pronounced influence of genotype-specific modifiers. However, the contribution of parthenogenetic cells to the brain does appear to be influenced by strain background, since a marked improvement in the survival of CFLP, 129 and perhaps SWR parthenogenetic cells in chimeric brains was observed compared with F2 cells.     

Developmental Potential of Haploid-derived Parthenogenetic Cells in Mouse Chimeric Embryos1

Studies were made on the contribution of haploid-derived parthenogenetic cells to haploid parthenogenetic ? fertilized chimeric embryos on day 9 and 10 of pregnancy. In most cases, the contribution of haploid-derived parthenogenetic cells to embryonic tissues was higher than that to extraembryonic tissues. The contribution of haploid-derived cells to embryonic tissues of some chimeras was more than 90%. Chromosomal analysis showed that actively dividing cells in most chimeric embryos contained about 40 chromosomes, indicating that they were diploidized, as haploid parthenogenetic blastocysts have about 20 chromosomes. Results suggested that haploid-derived parthehogenetic cells in chimeric embryos diploidized spontaneously after the blastocyst stage. These cells were capable of differentiating into most cell types of embryonic tissues, but scarcely differentiated into extraembryonic tissues of day 9 embryos. The fate of haploid-derived parthenogenetic cells during postimplantational development was similar to that of diploid parthenogenetic cells that had been diploidized experimentally in the one-cell stage.     

The development potential of parthenogenetically derived cells in chimeric mouse embryos: implications for action of imprinted genes

Parthenogenetic embryos of mice die shortly after implantation and characteristically contain poorly developed extraembryonic tissue. To investigate the basis of the abnormal development of parthenotes, we combined them with normal embryos to produce chimeras and examined the distribution of the parthenogenetically derived cells during preimplantation and early postimplantation development. The parthenogenetic embryos were derived from a transgenic mouse line bearing a large insert, which allowed these cells to be identified in histological sections using in situ hybridization. At the blastocyst stage, the parthenogenetic embryos contributed cells to the trophectoderm (TE) and inner cell mass (ICM) of chimeras. By 6.5 days, however, in almost every embryo, parthenogenetically derived cells were not detected in the extraembryonic trophoblast tissue descended from the TE. In contrast, parthenogenetically derived cells could contribute to all descendants of the ICM of 6.5-and 7.5-day chimeras, including the extraembryonic visceral and parietal endoderm. Quantitative analysis of the degree of chimerism in the embryonic ectoderm at 6.5-7.5 days indicated that parthenogenetically derived cells could contribute as extensively as normal cells. These results indicate that normal trophoblast development requires gene expression from the paternally inherited genome before 6.5 days of embryogenesis. Tissues of the ICM lineage, however, apparently can develop independently of the paternal genome at least to 7.5 days of embryogenesis. Comparison of these results with those of others suggests that the influence of imprinted genes is manifested at different times and in a variety of tissues during development.     

Chimeras between parthenogenetic or androgenetic blastomeres and normal embryos: allocation to the inner cell mass and trophectoderm

A series of chimeras was generated by injecting single normal, parthenogenetic, or androgenetic blastomeres carrying transgenic markers under the zona pellucida of nontransgenic eight-cell embryos. These chimeras were cultured to the blastocyst stage and sectioned, and the transgenic component was detected by in situ hybridization. No statistically significant difference was found among the normal, parthenogenetic, and androgenetic chimeras in the number of chimeric blastocysts with a transgenic contribution to the inner cell mass (ICM), the trophectoderm, or both the ICM and trophectoderm. Since androgenetic and parthenogenetic cells were present in chimeras at a high frequency in both the ICM and trophectoderm at the blastocyst stage, but not in similar chimeras at late gastrulation, these cells must not respond normally to developmental signals subsequent to blastocyst formation and prior to late gastrulation.     

Different influences of genomic imprinting on the development of parthenogenetic cell clones in C57BL/6 and CBA mice

Clonal analysis of parthenogenetic chimeric mouse embryos C57B1/6(PG)<-->BALB/c has shown that parthenogenetic cell clones C57BL/6 are present in the brain, liver, and kidneys of 14- and 18-day-old embryos. The content of the parthenogenetic component (PG) in these organs on day 18 was lower than on day 14, and, in some 18-day-old embryos, parthenogenetic cell clones were absent from the liver and/or kidneys. These data suggest that, during the embryogenesis of parthenogenetic chimeras, parthenogenetic cell clones of mostly endodermal and mesodermal origins were actively eliminated. Therefore, in such parthenogenetic adult chimeras, parthenogenetic clones of mostly ectodermal origins were preserved. In parthenogenetic chimeras CBA(PG)<-->BALB/c, parthenogenetic cell clones were actively eliminated at early embryonic stages, and, as a result, they were absent at the post-implantation stages. Hence, during development of parthenogenetic cell clones, the effects of genomic imprinting are expressed unequally in C57BL/6 and CBA mice.     

In vivo and in vitro differentiation of uniparental embryonic stem cells into hematopoietic and neural cell types

The biological role of genomic imprinting in adult tissue is central to the consideration of transplanting uniparental embryonic stem (ES) cell-derived tissues. We have recently shown that both maternal (parthenogenetic/gynogenetic) and paternal (androgenetic) uniparental ES cells can differentiate, both in vivo in chimeras and in vitro, into adult-repopulating hematopoietic stem and progenitor cells. This suggests that, at least in some tissues, the presence of two maternal or two paternal genomes does not interfere with stem cell function and tissue homeostasis in the adult. Here, we consider implications of the contribution of uniparental cells to hematopoiesis and to development of other organ systems, notably neural tissue for which consequences of genomic imprinting are associated with a known bias in development and behavioral disorders. Our findings so far indicate that there is little or no limit to the differentiation potential of uniparental ES cells outside the normal developmental paradigm. As a potentially donor MHC-matching source of tissue, uniparental transplants may provide not only a clinical resource but also a unique tool to investigate aspects of genomic imprinting in adults.Key words: uniparental, androgenetic, chimera, transplantation, parthenogenetic, gynogenetic, hematopoietic, neural     

Different Influences of Genomic Imprinting on the Development of Parthenogenetic Cell Clones in C57BL/6 and CBA Mice

Clonal analysis of parthenogenetic chimeric mouse embryos C57BL/6(PG) BALB/c has shown that parthenogenetic cell clones C57BL/6 are present in the brain, liver, and kidneys of 14- and 18-day-old embryos. The content of the parthenogenetic component (PG) in these organs on day 18 was lower than on day 14, and, in some 18-day-old embryos, parthenogenetic cell clones were absent from the liver and/or kidneys. These data suggest that, during the embryogenesis of parthenogenetic chimeras, parthenogenetic cell clones of mostly endodermal and mesodermal origins were actively eliminated. Therefore, in such parthenogenetic adult chimeras, parthenogenetic clones of mostly ectodermal origins were preserved. In parthenogenetic chimeras CBA(PG) BALB/c, parthenogenetic cell clones were actively eliminated at early embryonic stages, and, as a result, they were absent at the post-implantation stages. Hence, during development of parthenogenetic cell clones, the effects of genomic imprinting are expressed unequally in C57BL/6 and CBA mice.     

Systematic non-uniform distribution of parthenogenetic cells in adult mouse chimaeras

Even though pure parthenogenetic mouse embryos die shortly after implantation, their cells are capable of participating in normal development of chimaeras when aggregated with fertilized embryos. Here we present data on parthenogenetic contribution to the oocyte populations measured by progeny tests in female chimaeras, and on distribution of parthenogenetic cells among the different organs by GPI typing. Systematic uneven distribution was detected. The highest level of participation was registered in the tissues of permanent cells (e.g. up to 63% in female germline). On the other hand, parthenogenetic cells were absent in several tissues that have extensive capacity for postnatal growth or selfrenewal. This finding suggests that uneven selective processes operate against parthenogenetic cells within certain differentiation pathways during fetal and postnatal life, as has already been observed in the development of extraembryonal membranes. It is likely that more than one mechanism is responsible for these selections. Parthenogenetic cells may start to differentiate in all cell lineages, but they are not able to react normally at certain points in the developmental pathway, for example to induction signals and, therefore, the cells fail to complete the normal processes of development, or to the proliferation requirement so that the fertilized counterpart gradually takes over the cell lineage. Paternally derived gene(s) might have a unique role in the development of tissues lacking parthenogenetic contribution.     

The use of chimeric mice in studying the effects of genomic imprinting

This is a review of the data of clonal analysis of developing tissues in parthenogenetic and androgenetic chimeric mice. The time and causes of death of the parthenogenetic and androgenetic cell clones in chimeras are considered. The data obtained suggest that the development of cell clones, derivatives of the mesoderm and endoderm, is determined by the expression of alleles of the imprinted loci of paternal chromosomes, while the formation of cell clones, derivatives of the ectoderm, depends on the expression of other imprinted loci of maternal chromosomes. The death of androgenetic and parthenogenetic (gynogenetic) mammalian embryos is due to the lack of the expression of certain imprinted loci of the maternal and paternal genome, respectively.     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

Use of chimeric mice for studying the effects of genomic imprinting

This is a review of the data of clonal analysis of developing tissues in parthenogenetic and androgenetic chimeric mice. The time and causes of death of the parthenogenetic and androgenetic cell clones in chimeras are considered. The data obtained suggest that the development of cell clones, derivatives of the mesoderm and endoderm, is determined by the expression of alleles of the imprinted loci of paternal chromosomes, while the formation of cell clones, derivatives of the ectoderm, depends on the expression of other imprinted loci of maternal chromosomes. The death of androgenetic and parthenogenetic (gynogenetic) mammalian embryos is due to the lack of the expression of certain imprinted loci of the maternal and paternal genome, respectively.     

Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting

The inviability of diploid androgenetic and parthenogenetic embryos suggests imprinting of paternal and maternal genes during germ cell development, and differential expression of loci depending on parental inheritance appears to be involved. To facilitate identification of imprinted genes, we have derived diploid androgenetic embryonic stem (ES) cell lines. In contrast to normal ES cells, they form tumors composed almost entirely of striated muscle when injected subcutaneously into adult mice. They also form chimeras following blastocyst injection, although many chimeras die at early postnatal stages. Surviving chimeras develop skeletal abnormalities, particularly in the rib cartilage. These results demonstrate that androgenetic ES cells are pluripotent and point to stage- and cell-specific expression of developmentally important imprinted genes.     

Production and properties of mouse trisomy 15 ⟷ diploid chimeras

Mouse trisomy 15 ? 2n aggregation chimeras have been produced and analyzed at 19 days of gestation. We have found that these chimeras are viable and in most instances normal in external appearance, unlike trisomy (Ts)-15 embryos which are severely growthretarded and die midway through gestation. Trisomic cells were found in all tissues of fetal chimeras, with proportions not significantly different from those of the controls in kidney, heart, liver, and brain, but significantly reduced in thymus and spleen. Ts-15 cells do not, therefore, exhibit a proliferative advantage during fetal development of tissues susceptible to Ts-15-related lymphoid malignancies. However, the presence of Ts-15 cells in the placenta may be associated with placental overgrowth. One fetus containing a monosomy 3 cell population was also observed, the first term fetal chimera with monosomic cells that has been detected.     

Parthenogenetic stem cells in postnatal mouse chimeras.

The ability of parthenogenetic (pg) cells to contribute to proliferating stem cell populations of postnatal aggregation chimeras was investigated. Using DNA in situ analysis, pg participation was observed in highly regenerative epithelia of various regions of the gastrointestinal tract, e.g., stomach, duodenum and colon, in the epithelia of tongue and uterus and in the epidermis. Pg cells also contributed to the epithelium of the urinary bladder, which is characterized by a relatively slow cellular turnover. Using a sensitive proliferation marker to determine division rate of pg and normal (wt) cells in tissues of a 24-day-old chimera, no significant differences between pg and fertilized cells were observed. However, in colon and uterus of a pg <==> wt chimera aged 101 days, a significant loss of proliferative capacity of pg cells was found. In the colon, this loss of proliferative potential was accompanied by an altered morphology of pg crypts. In general, they were situated at the periphery of the epithelium and lacked access to the lumen, with consequent cystic enlargement and flattened epithelium. No obvious morphological changes were observed in the pg-derived areas of the uterine epithelium of this chimera. Our results provide evidence that pg cells can persist as proliferating stem cells in various tissues of early postnatal chimeras. They suggest that pg-derived stem cells may cease to proliferate in restricted areas of the gastrointestinal tract and in the uterine epithelium of pg <==> wt chimeras of advanced age.(ABSTRACT TRUNCATED AT 250 WORDS)