Temporal and spatial selection against ... - Semantic Scholar

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Development 108,203-211 (1990) Printed in Great Britain © The Company of Biologists Limited 1990

Temporal and spatial selection against parthenogenetic cells during development of fetal chimeras REINALD H. FUNDELE1, MICHAEL L. NORRIS1, SHEILA C. BARTON1, MONIKA FEHLAU2, SARAH K. HOWLETT1, WALTER E. MILLS1 and M. AZIM SURANI1 1 2

Department of Molecular Embryology, AFRC Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, UK Institut fiir Physiologische Chemie, Physikalische Biochemie und Zellbiologie der Universitdt, Goethestr. 33, 8000 MUnchen 2, FRG

Summary 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«->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 deceleration of parthenogeneticfertilized 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 parthenogeneticfertilized chimeras.

Introduction

tiated cell types (lies etal. 1975). Extensive studies have also shown that, while parthenogenetic cells are detected in adult chimeras (Stevens, 1978; Stevens etal. 1977; Surani et al. 1977; Anderegg and Markert, 1986; Otani et al. 1987; Surani et al 1987; Surani et al. 1988), they are subject to strong selective pressure in the conceptus. Although the initial allocation of parthenogenetic cells in preimplantation chimeric embryos is apparently random (Clarke et al. 1988; Thomson and Solter, 1989), these cells are progressively eliminated, first from the trophoblast, followed by yolk sac endoderm and then yolk sac mesoderm. By midgestation, parthenogenetic cells are virtually absent from the extraembryonic tissues (Nagy et al. 1987; Surani et al. 1988; Clarke etal. 1988; Thomson and Solter, 1988). In the fetus, these cells continue to proliferate normally until midgestation (Nagy et al. 1987; Surani et al. 1988). Thereafter, there is an overall decrease in the contribution of parthenogenetic cells to the embryo as well

The two parental genomes of the mouse are not functionally equivalent (Cattanach, 1986; Surani et al. 1986). The mechanism that confers functional differences on parental genomes has been called genomic imprinting (Surani et al. 1984), by analogy with the phenomenon of preferential paternal X chromosome inactivation in extraembryonic tissues of the mouse (Tagaki and Sasaki, 1975; Lyon and Rastan, 1984). Both androgenesis (two paternal chromosome sets) and gynogenesis/parthenogenesis (two maternal chromosome sets) result in prenatal lethality but result in opposite phenotypes. (McGrath and Solter, 1984; Surani et al. 1984). It has previously been shown that parthenogenetic (gynogenetic) embryos can give rise to pluripotential embryonic stem cells (Robertson et al. 1983) as well as to teratomas in ectopic sites with a variety of differen-

Key words: parthenogenesis, fetal chimeras, genomic imprinting, cell selection/proliferation.

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R. H. Fundele and others

(Nagy et al. 1987). Nevertheless, in adult chimeras parthenogenetic cells may be present in most tissues, including the germ cells (Stevens, 1978; Stevens et al. 1977; Surani et al. 1977; Anderegg and Markert, 1986; Otani et al. 1987; Paldi et al. 1989; Fundele et al. 1989; Nagy et al. 1989). Selection against parthenogenetic cells does not appear to occur uniformly, as in some tissues these cells are detected much more consistently than in others (Fundele et al. 1989; Nagy et al. 1989). The aim of this study was to determine in greater detail the onset of selective elimination of parthenogenetic cells in different tissues of fetal chimeras. Furthermore, various stages of gestation were analysed in order to determine the timecourse of their elimination. This study establishes a basis for further analysis of the mechanisms of cell selection in parthenogeneticfertilized chimeras and provides further insight into the influence of parental chromosomes on development. Materials and methods Animals Outbred albino CFLP mice (Gpi-la/Gpi-la) and nonalbino (C57BL/6JxCBA/Ca)Fi mice (Gpi-lh/Gpi-lb), originally from Bantin and Kingman stock, were used. Female mice were superovulated according to standard procedures (Fowler and Edwards, 1957). Embryos Normal fertilized embryos were obtained after in vivo fertilization from F ( xF! or CFLPxCFLP matings. For the production of parthenogenetic embryos, oocytes from F! females were collected at 17.5-19.0 h after injection of human chorionic gonadotrophin (Intervet Ltd., Cambridge). The cumulus cells were removed by incubation with 300i.u. ml"1 hyaluronidase (ovine testis type V, Sigma) in PB1+BSA (Whittingham and Wales, 1969) for 1-2 min and washed through 3 drops of medium T6+BSA (Howlett et al. 1987). The eggs were activated in T6+BSA containing 7 % ethanol for 4.5 min at room temperature (Kaufman, 1982; Cuthbertson, 1983), washed six times with T6+BSA and cultured in this medium with 5/igmF 1 cytochalasin B (0.1% in dimethylsulphoxide) for 3.5-4 h at 37.8°C in 5% CO2 in air. The eggs were then washed seven times in T6+BSA and cultured for a further period of 2 h at 37.8°C in 5 % CO2 in air. After this time all the diploid eggs containing two pronuclei (70-90 %) resulting from prevention of the second meiotic division by cytochalasin B (Niemierko, 1975) were separated from haploid, fragmented and other abnormal eggs and cultured for 2 days in T6+BSA. Production of aggregation chimeras Asynchronous aggregation chimeras were made by combining 4-cell embryos, either fertilized (FiXF!)F2 or parthenogenetic, with 2-cell fertilized CFLP embryos. Synchronous aggregation chimeras to serve as controls were prepared with 4-cell embryos. The methods used in our laboratory for the production of aggregation chimeras have been fully described previously (Surani et al. 1988; Fundele et al. 1989). For the production of some of the adult chimeras, parthenogenetic inner cell masses obtained by immunosurgery (Solter and Knowles, 1975) on day 5 of development were injected into day 4 (CFLPxCFLP) blastocysts. Blastocysts from aggregated embryos were transferred to recipient females on day 3

of pseudo-pregnancy, irrespective of the age of aggregated embryos. No control embryos were transferred with the aggregated or injected embryos. The recipient females were obtained by mating mature females with vasectomized males of proven sterility. Pregnant recipient females were killed by cervical dislocation on different days after embryo transfer and the fetuses removed. Alternatively, females were left until term on day 20 (=postnatal day 1; al). Fetal chimeras were exsanguinated by opening the hepatic vein in order to minimize contamination of tissues with blood. Postnatal chimeras were first injected intraperitoneally with approximately 700 units of heparin 30 min before killing followed by exsanguination. Whole organs were collected and homogenized for GPIanalysis on fetal days 13 and 15 (el3, el5) and, in the case of most tissues, on all other fetal and postnatal days. Only tongue was analyzed on el3 and el5, as it was found impossible to dissect other defined skeletal muscles at these developmental stages. However, skeletal muscle samples were taken from several sites from day 17 of gestation onwards. Similarly, three different regions of the brain and liver were collected from day 17 onwards. Bodyweights of parthenogeneticfertilized chimeras and control CFLP littermates that had lost the parthenogenetic F2 contribution were determined after removal of the fetal membranes. For an analysis of size difference between F 2 and CFLP fetuses at different developmental stages, day 4 F2 and CFLP blastocysts were transferred together to day 3 pseudopregnant F ( females. Nine adult female parthenogenetic