Fate of embryonal carcinoma cells injected into postimplantation mouse embryos

Embryonal carcinoma (EC) cells, stem cells of teratocarcinoma, represent an excellent model to study the developmental mechanisms that, inappropriately reactivated, can drive tumorigenesis. EC cells are very aggressive, and grow rapidly when injected into adult syngeneic mice. However, when injected into blastocysts, they revert to normality, giving rise to chimeric animals. In order to study the ability of postimplantation embryonic environment to "normalize" tumorigenic cells, and to study their homing, we transplanted F9, Nulli-SCC1, and P19 EC cells into 8 to 15-day allogenic CD1 mouse embryos, into allogenic CD1 newborns, and into syngeneic adult mice, and evaluated tumor formation, spreading, and homing. We found that, although at all embryonic stages successful transplantation occurred, the chances of developing tumors after birth increased with the time of injection of EC cells into the embryo. In addition, using enhanced green fluorescent protein-expressing F9 cells, we demonstrated that the cells not giving rise to tumors remained latent and could be tracked down in tissues during adulthood. Our data indicate that the embryonic environment retains a certain ability to "normalize" tumor cells also during post-implantation development. This could occur through yet unknown epigenetic signals triggering EC cells' differentiation.     

On the fate of primordial germ cells injected into early mouse embryos

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells – pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs.     

Interspecific melanocyte chimaeras made by introducing rat cells into postimplantation mouse embryos in utero

Dissociated cells of whole midgestation rat embryos were injected into implanted albino mouse embryos on Day 8.5 of gestation in utero. This successfully produced viable interspecific chimaeras which were found to have pigmented hairs. Two of them had many pigmented hairs covering a large area of their bodies, including a forelimb and a hindlimb. The fact that some of the introduced rat cells differentiated into functional melanocytes suggests that embryonic cells of both species were able to interact with each other normally and that the foreign cells were kept from maternal immunological assault.     

Effect of nicergoline and dihydroergotamine, injected into the cerebral ventricles, on arterial pressure in the cat

Nicergoline, injected into the cerebral ventricles of anaesthetized cats induced a moderate, but longlasting and dose-dependent fall of the blood pressure. Dihydroergotamine, in moderate doses, had no effect on the arterial blood pressure. Toxic doses of nicergoline and dihydroergotamine provoked a pronounced fall of the arterial blood pressure and inhibition of the respiration. Bradycardia only appeared after toxic doses of dihydroergotamine. It is concluded that the hypotensive effect of nicergoline is at least in part central in origin.     

Apolipoprotein e deficiency leads to altered brain uptake of alpha tocopherol injected into lateral cerebral ventricles

The incorporation of radioactive alpha tocopherol by various brain regions of wild type and apolipoprotein E (apoE)-deficient mice was investigated. Labeled tocopherol was injected into the lateral cerebral ventricles of 11 weeks old, male mice. Radioactive cholesterol injected simultaneously was used as an internal standard to account for experimental variability. Most areas of the brain of apoE-deficient mice took up less of alpha tocopherol per mg of protein than wild type animals. However, specific activity of alpha tocopherol was higher in cerebellum, pons, hypothalamus, midbrain and cerebral cortex in apoE-deficient brains than the wild type. This could be due to (a) the lower levels of alpha tocopherol in apoE-deficient brain and (b) reductions in the clearance and transport of tocopherol (possibly mediated by apoE). Tocopherol uptake by hippocampus was unusual since it was lower in apoE deficiency whether the data were expressed as specific activity or per mg of protein. Nearly all of the injected alpha tocopherol remained unchanged in the brains of both apoE-deficient and wild type animals suggesting low turnover. Overall, the current data reinforce the hypothesis that apoE is a key protein involved with the transport and/or retention of alpha tocopherol in brain.     

Feeding induced by opiates injected into the paraventricular hypothalamus

Injection of morphine or d-ala-2-met-5-enkephalinamide (DALA) into the paraventricular nucleus of the hypothalamus (PVN) produced a dose dependent increase in feeding in rats. DALA increased feeding within 45 minutes; morphine within 90 minutes. Naloxone injected into the PVN diminished the effect. DALA increased water intake only when food was available, suggesting the primary effect was on feeding. In summary, an enkephalin analogue induced feeding, and an opiate receptor blocker attenuated it; therefore the PVN may contain opiate receptors that facilitate feeding.     

A comparison of the central and peripheral antimuscarinic effects of atropine and methylatropine injected systemically and into the cerebral ventricles

We compared the relative abilities of atropine sulfate and methylatropine, injected i.v. and into the cerebral ventricles (icv), to block pharmacological responses mediated through central and peripheral muscarinic receptors. The hypotensive response to i.v. injection of acetylcholine (peripheral muscarinic receptors) was inhibited 50% by i.v. injection of 14.3 nmol (5.5 micrograms)/kg methylatropine and 147.8n molar equivalents (50 micrograms)/kg atropine sulfate. A similar degree of inhibition followed icv injection of 49.4 nmol/kg methylatropine and 384.2 nmol equivalents/kg atropine sulfate, indicating significant leakage out of the ventricular space. The pressor response to icv injection of neostigmine (central muscarinic receptors) also was inhibited more effectively by icv methylatropine than by atropine sulfate. Methylatropine was not effective in blocking central muscarinic receptors when injected i.v.     

Sonographic staging of the developmental status of mouse embryos in utero

In mouse developmental studies it is frequently desirable to isolate embryos of a specific age. However, the traditional staging of embryonic development based on postcoital dates often erroneously predicts the embryonic age, resulting in unwarranted sacrifice of the pregnant mother. Here we report a noninvasive way of staging embryonic development in utero. A clinical 14 MHz ultrasound system was employed to assess the morphology and size of developing embryos from embryonic day 7.5 to 18.5. We demonstrate that the developmental age of the mouse embryos can be accurately determined based on the sonographic morphology and size of the embryos. This noninvasive ultrasound application requires no anesthesia of the mice and the entire process of staging can be completed within 5-10 min. Empirically, this approach is applicable to mice of various genetic backgrounds and significantly enhances the efficiency of studying murine embryogenesis.     

Fate of DNA injected into early Drosophila embryos

Plasmid DNA injected into early Drosophila embryos becomes enclosed in nuclei or nucleus-like structures where it remains at least throughout embryonic development. The fraction remaining in the cytoplasm is gradually degraded. The nuclear fraction is converted to a high-molecular-weight form consisting largely but not exclusively of tandem oligomers. Some of it, however, can occasionally become integrated in the genome. Extensive DNA replication takes place but few, if any, molecules are able to initiate a second round of replication.     

Replication analysis of plasmid DNAs injected into Drosophila embryos

From a shotgun collection of DNA fragments, isolated from Drosophila melanogaster, we selected sequences that function as autonomously replicating sequences (ARS) in the yeast Saccharomyces cerevisiae. To investigate the replicative potential of such sequences in Drosophila, five of these ARS elements and also the Adh gene of D. melanogaster, which has been described earlier to have ARS function in yeast, were microinjected into developing Drosophila eggs and analysed after reisolation from first instar larvae. As an assay for DNA replication, we determined the sensitivity of recovered plasmid DNA to restriction enzymes that discriminate between adenine methylation and nonmethylation. within the limits of detection our results show that none of the plasmids replicated two or more rounds. However, a fraction of all injected plasmid DNAs, including vector DNA, seems to replicate once. The same result was obtained for a DNA sequence from mouse that had been reported to have replication origin function in mouse tissue culture cells. We excluded the possibility that methylation of the plasmids is the reason for their inability to replicate. These results demonstrate that homologous and heterologous DNA sequences that drive replication of plasmids in cells of other species are not sufficient to fulfil this function in Drosophila embryos.by J.A. Huberman     

Tissue-specific expression of actin genes injected into Xenopus embryos

We have isolated a complete Xenopus borealis cardiac actin gene, which is normally expressed in the myotomes and heart of the embryo and tadpole. After injection into the zygote, this cloned gene becomes distributed throughout the embryo, but it is expressed almost wholly in the myotomes. The same wide distribution of injected DNA but spatially restricted pattern of expression is found with a fusion between the first two actin gene exons and the last exon of a mouse beta-globin gene. By contrast, a histone-globin fusion gene is expressed fairly uniformly in all regions. We discuss the special advantages of using Xenopus in studies of tissue-specific gene expression from injected, cloned genes in early development.