Christopher F. Graham

Changes in the cell cycle during early amphibian development

The time for which a cell is in each phase of the cell cycle, during the early development of a limited region of the endoderm in Xenopus laevis, has been calculated using 3H-thymidine as a specific marker of DNA synthesis. Labeled thymidine was injected into the embryo, and its subsequent incorporation into the cell nuclei was observed. A mathematical model is presented for calculating the phases of the cell cycle in any system in which the length of the cell cycle and its phases are changing. During cleavage there is no G1 phase and the G2 phase is either very short or absent. In the late blastula there is a substantial G2 phase and the G1 phase becomes apparent. From the late blastula to the early tail bud stage, the duration of the G1, S, and G2 phases increase but the duration of the M phase remains relatively constant. The lengths of all the phases of the cell cycle are summarized in Fig. 8.

Appearance of interferon inducibility and sensitivity during differentiation of murine teratocarcinoma cells in vitro

Pluripotential embryonal carcinoma (EC) cells do not produce interferon after treatment with a wide variety of inducers, nor are they sensitive to its action. Several differentiated lines derived from the EC cells, however, both produce and are sensitive to mouse interferon. Differentiation of EC cells in vitro is accompanied by development of interferon inducibility and sensitivity.

The differentiation of teratocarcinoma stem cells is marked by the types of collagen which are synthesized

Abstract A cloned line of undifferentiated teratocarcinoma cells (OC15S1) was either maintained as a homogeneous embryonal carcinoma (EC) cell population or was cultured under conditions where the cells differentiated into endoderm-like (END) cells. In this study we examine the synthesis of collagen in both EC and END cells. Cell cultures were incubated with tritiated proline and lysine, and the radioactive collagen secreted into the medium was extracted and purified or immunoprecipitated by antibodies to type IV collagen (Adamson and Ayers, 1979). Radioactive collagens were identified by electrophoretic mobility, by sensitivity to collagenase and to reduction, by insensitivity to pepsin, by cyanogen bromide peptides, and by aminoacid analyses of 3-hydroxyproline, 4-hydroxyproline and proline. OC15S1 EC cells were found to synthesize several collagenous polypeptides, of which 60–70% of the radioactivity was like that of basement membrane (type IV) collagen. Type I-like collagen was the main collagenous product of END cells, but a minor product of EC cells. We concluded that type IV collagen synthesis was suppressed during the differentiation of EC cells to END, while type I-like synthesis was increased. Similarly, other EC cell lines produced mainly type IV-like collagen polypeptides (PC13, F9, PSA1), and following the formation of END cells, two lines produced mainly type I-like collagen polypeptides (PC13, C145b). The type of endoderm formed on embryoid bodies, however, presents an alternate route of differentiation, since immunoperoxidase tests showed that it was synthesizing significant amounts of type IV collagen. We discuss the significance of these findings in relation to a similar change which occurs during normal development.

Maternally-inherited Grb10 reduces placental size and efficiency.

The control of foetal growth is poorly understood and yet it is critically important that at birth the body has attained appropriate size and proportions. Growth and survival of the mammalian foetus is dependent upon a functional placenta throughout most of gestation. A few genes are known that influence both foetal and placental growth and might therefore coordinate growth of the conceptus, including the imprinted Igf2 and Grb10 genes. Grb10 encodes a signalling adapter protein, is expressed predominantly from the maternally-inherited allele and acts to restrict foetal and placental growth. Here, we show that following disruption of the maternal allele in mice, the labyrinthine volume was increased in a manner consistent with a cell-autonomous function of Grb10 and the enlarged placenta was more efficient in supporting foetal growth. Thus, Grb10 is the first example of a gene that acts to limit placental size and efficiency. In addition, we found that females inheriting a mutant Grb10 allele from their mother had larger litters and smaller offspring than those inheriting a mutant allele from their father. This grandparental effect suggests Grb10 can influence reproductive strategy through the allocation of maternal resources such that offspring number is offset against size.

Enolase isoenzymes as markers of differentiation in teratocarcinoma cells and normal tissues of mouse.

Abstract The changing profile of enolase (EC 4.2.1.11) isoenzymes in differentiating mouse cells has been traced by the use of specific antisera to the three subunits α, β, and γ. The amounts of the isoenzymes were measured in a variety of tissues during normal mouse development and during the differentiation of mouse teratocarcinoma cells in culture and as tumors. One isoenzyme is predominant in the early cells of the developing mouse embryo, namely, the homodimer made up of α subunits. The same isoenzyme is also the sole form detected in undifferentiated teratocarcinoma (embryonal carcinoma) cells. The isoenzyme form remains unchanged in developing primitive and definitive endoderm of the embryo. Similarly, endoderm cells formed by differentiation of embryonal carcinoma cells contained only αα enolase. In contrast, during the development of striated muscle and of brain, increasing proportions of β and γ subunits, respectively, were detected. Thus enolase was found to be a marker of the differentiation of these tissues. This conclusion was substantiated by finding significant amounts of the β subunit in teratocarcinoma cell cultures which had formed beating striated muscle in vitro .

Phospholipase C-ζ-induced Ca2+ oscillations cause coincident cytoplasmic movements in human oocytes that failed to fertilize after intracytoplasmic sperm injection

Objective To evaluate the imaging of cytoplasmic movements in human oocytes as a potential method to monitor the pattern of Ca2+ oscillations during activation. Design Test of a laboratory technique. Setting University medical school research laboratory. Patient(s) Donated unfertilized human oocytes from intracytoplasmic sperm injection (ICSI) cycles. Intervention(s) Microinjection of oocytes with phospholipase C (PLC) zeta (ζ) cRNA and a Ca2+-sensitive fluorescent dye. Main Outcome Measure(s) Simultaneous detection of oocyte cytoplasmic movements using particle image velocimetry (PIV) and of Ca2+ oscillations using a Ca2+-sensitive fluorescent dye. Result(s) Microinjection of PLCζ cRNA into human oocytes that had failed to fertilize after ICSI resulted in the appearance of prolonged Ca2+ oscillations. Each transient Ca2+ concentration change was accompanied by a small coordinated movement of the cytoplasm that could be detected using PIV analysis. Conclusion(s) The occurrence and frequency of cytoplasmic Ca2+ oscillations, a critical parameter in activating human zygotes, can be monitored by PIV analysis of cytoplasmic movements. This simple method provides a novel, noninvasive approach to determine in real time the occurrence and frequency of Ca2+ oscillations in human zygotes.

Neuronal differentiation of cloned human teratoma cells in response to retinoic acid in vitro.

We have analysed immunocytochemically the differentiation in vitro of clones of the human teratoma cell line Tera-2. The proliferating stem cells expressed Thy-1 and N-CAM/D2-CAM antigens. On treatment with 5 X 10(-5) M retinoic acid in either monolayer or aggregate cultures they began to express receptors for tetanus toxin and McAb A2B5. Three weeks after initiating retinoic acid treatment, the cultures contained a variety of cell morphologies, including about 3% of cells with a neuron-like morphology. These cells were reactive with tetanus toxin, McAb A2B5, and antibodies against Thy-1 and N-CAM/D2-CAM. They also expressed 55 000 and 210 000 Da neurofilament subunits.

Genetic Manipulation of Mouse Embryos

Summary This paper reviews the most recent attempts to introduce genetic material into the developing mouse embryo. First, attempts have been made to suppress second polar body formation to obtain parthenogenetic diploid embryos. This manipulation has been performed successfully but the embryos do not develop beyond the seventh day of pregnancy. Secondly, attempts have been made to fuse adult cell nuclei into fertilized and unfertilized eggs. These additional nuclei undergo chromosome condensation, and chromosome fragmentation. However, unlike somatic cell hybrids, the fragmented genome does not appear to be incorporated into the developing embryos. However, embryos exposed to these fusion conditions develop into live young, showing that fusion itself does not block the development of mammalian embryos.

Igf2 pathway dependency of the Trp53 developmental and tumour phenotypes

Insulin‐like growth factor 2 (IGF2) and the transformation related protein 53 (Trp53) are potent regulators of cell growth and metabolism in development and cancer. In vitro evidence suggests several mechanistic pathway interactions. Here, we tested whether loss of function of p53 leads to IGF2 ligand pathway dependency in vivo. Developmental lethality occurred in p53 homozygote null mice that lacked the paternal expressed allele of imprinted Igf2. Further lethality due to post‐natal lung haemorrhage occurred in female progeny with Igf2 paternal null allele only if derived from double heterozygote null fathers, and was associated with a specific gene expression signature. Conditional deletion of Igf2fl/fl attenuated the rapid tumour onset promoted by homozygous deletion of p53fl/fl. Accelerated carcinoma and sarcoma tumour formation in p53+/− females with bi‐allelic Igf2 expression was associated with reductions in p53 loss of heterozygosity and apoptosis. Igf2 genetic dependency of the p53 null phenotype during development and tumour formation suggests that targeting the IGF2 pathway may be useful in the prevention and treatment of human tumours with a disrupted Trp53 pathway.