Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.     

Modification of survival rate of mouse embryos developing in heterozygous females for ovum mutant gene

The DDK syndrome (polar infertility) is caused by an incompatibility system due to the ovum mutant (Om) locus. For brevity, the following gene symbols are used in the present report: DDK allele, Om; C57BL/6Cr allele, +. In this investigation, we first attempted to introduce the Om allele of DDK strain into the genetic background of C57BL/6Cr strain. The attempt resulted in the production of no young at the third generation of successive backcrosses. Secondly, mating experiments were performed with heterozygous (Om/+) females having background genes of C57BL/6Cr and DDK strains in the ratios 1:1(B1D), 3:1(B3D), 7:1(B7D), and 15:1(B15D). The survival rate of the embryos as judged by the percentage number of live fetuses/number of corpora lutea at Day 12 of pregnancy was 41.3 +/- 3.2%, 27.3 +/- 3. 2%, 16.4 +/- 3.3%, and 11.3 +/- 3.2% (mean +/- SEM) in the B1D, B3D, B7D, and B15D females, respectively, when they were mated with C57BL/6Cr males. Furthermore, the increased embryonic mortality in the heterozygous (Om/+) females with more background genes of C57BL/6Cr strain was found to be due to a failure in blastocyst formation, as in the DDK syndrome. The parallelism between the proportion of C57BL/6Cr background genes and embryonic mortality has led to a hypothesis proposing the participation of a modifier gene, namely that a mechanism similar to allelic exclusion may be working in the synthesis of cytoplasmic factor of eggs and that only the Om allele is activated during oogenesis to produce DDK-type cytoplasmic factor in heterozygous (Om/+) females having a modifier gene in the homozygous state.     

An automated procedure to measure DNA synthesis in preimplantation mouse embryos

This paper describes a sensitive, reproducible, and automated procedure to measure DNA synthesis in preimplantation mouse embryos. Conditions for the DNA synthesis assay have been optimized as follows: (1) 4 μCi/ml³H-thymidine (sp. act. 20 Ci/immole); (2) a labeling period from 2 to 7 hours; (3) a 3-hour preincubation period for blastocysts and from 0 to 7 hours preincubation for 8-cell embryos; and (4) from 1 to 64 embryos per assay. The amount of DNA synthesis per embryo was found to be directly proportional to the number of cells (nuclei) per embryo. The described assay should be useful for future studies on the effect of synthetic and natural compounds on the development of preimplantation mouse embryos, as measured by perturbations in embryonic DNA synthetic activity.     

Enhanced susceptibility of mouse embryos heterozygous for oligosyndactyly (Os/+) to mitomycin C-induced skeletal abnormalities

The mouse mutation, oligosyndactyly (Os), results in syndactyly, muscle anomalies, and deficiency of nephrons in heterozygous animals and early embryonic lethality in homozygotes. Since the homozygous lethality results from mitotic arrest with intact spindles at the time of implantation, we have hypothesized that the heterozygous manifestations may result from impairment of cell proliferation in regions with high proliferative rates. To test this hypothesis, Os/+ and +/+ mouse embryos at 6.5 days of gestation were exposed to mitomycin C (MMC), an agent that causes a high degree of embryonic cell death which is "compensated" for by a period of rapid cell proliferation. 17.9% of MMC-treated +/+ fetuses had fused vertebrae, a significant increase over untreated fetuses, and this frequency was further increased to 33.6% in MMC-treated Os/+ fetuses. Saline treated Os/+ and +/+ fetuses showed the same low rate (0-3%) of vertebral fusion. These results indicate that Os/+ embryos have an increased sensitivity to the vertebral fusion-inducing effect of MMC at 6.5 days of gestation, a finding compatible with the hypothesis that rapid cell proliferation may be impaired in Os/+ embryos and fetuses.     

The synthesis and isolation of DNA complementary to histone mRNA from mouse embryos

A 9S poly(A)-RNA preparation isolated from mouse embryos was shown to stimulate the synthesis of histones in an ascites cell free extract. This RNA preparation was used for the synthesis of a highly labelled cDNA probe complementary to histone mRNA. Hybridisation of this cDNA probe to rRNA showed that 52% of the cDNA consisted of sequences complementary to rRNA. The histone mRNA specific cDNA was purified by hybridising the impure cDNA to rRNA followed by removal of the single-stranded histone cDNA by hydroxylapatite chromatography.     

Poly(ADP-ribose) polymerase activity in mouse cells which exhibit temperature-sensitive DNA synthesis

The poly(ADP-ribose) polymerase activity of wild-type mouse L cells and of Balb/C-3T3 mouse fibroblasts remained relatively unchanged (at approx. 400 nmol substrate utilized/mg DNA per h) in actively-growing cells incubated at 34 degrees C or at 38.5 degrees C for at least 72 h. A similar result was obtained with the following temperature-sensitive cells grown at the permissive temperature (34 degrees C): ts A1S9 mouse L cells, ts C1 mouse L cells and Balb/C-3T3 ts mouse fibroblasts. The poly(ADP-ribose) polymerase activity of the temperature-sensitive cells was little affected during incubation for 20-24 h at the non-permissive temperature of 38.5 degrees C under which conditions temperature-inactivation of DNA replication was complete. Thereafter, this enzyme activity was found to increase some 2-fold, at a time when normal semi-conservative DNA synthesis was totally suppressed and replaced by repair replication (Sheinin, R. and Guttman, S. (1977) Biochim. Biophys. Acta 479, 105-118; Sheinin, R., Dardick, I. and Doane, F.W. (1980) Exp. Cell. Res., in the press).     

Early protein synthesis during germination of barley embryos and its relationship to RNA synthesis

In barley embryo, protein synthesis as judged from the incorporationof labelled precursors, starts at about 15 min after the commencementof germination. Evidence suggests that these early proteinsare essential for germination and are programmed by a conservedpolyadenylate-containing mRNA, preserved in dry embryos. Althoughlow DNA-dependent RNA polymerase activity is present in drybarley embryos, RNA synthesis does not commence immediatelyafter water imbibition. It is initiated only after 2 hr of germinationand its synthesis requires the presence of early proteins. Furthermore,the activity of RNA polymerase increases with an increase ingermination time and after 40 hr of germination, the activityof RNA polymerase is about fivefold higher than that in dryembryo. However, cydoheximide completely blocks the enhancedactivity of RNA polymerase, suggesting a role of early proteinsin the initiation of new RNA synthesis in this developmentalsystem. (Received October 26, 1979; )     

Effects of ultrasound on DNA and RNA synthesis in preimplantation mouse embryos.

Ultrasound is used extensively to monitor the growth of ovarian follicles in in vitro fertilization and embryo transfer (IVF-ET) programs, as well as to follow the progress of early pregnancy. There have been scattered reports in the literature that exposure to ultrasound may have an adverse effect on reproduction in the rat (Bologne et al: CR Soc Biol 177:381-387, 1983; Demoulin et al: Ann NY Acad Sci 442:146-152, 1985), and also in humans (Demoulin et al: Ann NY Acad Sci 442: 146-152, 1985). We report here that diagnostic levels of pulsed ultrasound did not affect either the number of embryos produced, or the ability to incorporate labelled precursors into DNA and RNA, respectively. Measurements of temperature elevation of ovaries exposed to ultrasound showed that neither controls nor experimental tissue exhibited temperature elevation greater than 1 degree C.     

Lack of RNA-DNA oligonucleotide (chimeraplast) mutagenic activity in mouse embryos

There are numerous reports of the use of RNA-DNA oligonucleotides (chimeraplasts) to correct point mutations in vitro and in vivo, including the human apolipoprotein E gene (ApoE). Despite the absence of selection for targeting, high efficiency conversion has been reported. Although mainly used to revert deleterious mutations for gene therapy applications, successful use of this approach would have the potential to greatly facilitate the production of defined mutations in mice and other species. We have attempted to create a point mutation in the mouse ApoE gene by microinjection of chimeraplast into the pronuclei of 1-cell mouse eggs. Following transfer of microinjected eggs we analysed 139 E12.5 embryos, but obtained no evidence for successful conversion.     

Shifts in ATP synthesis during preimplantation stages of mouse embryos.

The actual and potential activities of the cyochrome system were studied in cleavage-stage mouse embroys. Activities were determined by assaying embroys for total ATP and the rates of [32-P]ATP synthesis both before and after their incubation in medium supplemented either with an energy coupling site inhibitor (antimycin, amytal or cyanide) or with the FADH-linked substrate, succinate. The data indicate that there are three major shifts in the mode of ATP production during preimplantation stages: the first, between the two-cell and late four-cell stages; the second, between the eight-celland late morula stages; and the third, between the late morula and late blastocyst stages. These data are discussed in relation to studies on the energy metabolism of cleavage and blastocyst stage mouse embryos.     

Elevated mutation rate in mutT bacteria during starvation: evidence for DNA turnover?

The rate of appearance of prototrophic revertants when Escherichia coli tyrA14 (ochre) or trpA23 bacteria were incubated on plates lacking the required amino acid was greatly elevated when the organisms also carried a mutT mutation. One possible explanation for this result is that the amount of DNA replication or turnover under these conditions is much greater than has been previously recognized.     

Expression of injected HPRT minigene DNA in mouse embryos and its inhibition by antisense DNA

We have used a highly sensitive biochemical microassay to monitor the expression of a cloned minigene for hypoxanthine phosphoribosyl transferase (HPRT, EC.2.4.2.8) in preimplantation mouse embryos. The mouse HPRT promoter and the mouse metallothionein promoter (MT-I) function equally well in embryos at the 2-cell stage whereas the viral SV40 promoter does not allow HPRT expression. Induced HPRT activity from the MT-I HPRT minigene construct occurs in cleavage embryos cultured in the presence of cadmium. In contrast, negation of enzyme expression from the injected minigene DNA is mediated by simultaneous injection of HPRT antisense DNA.     

The correlation of DNA synthesis and DNA polymerase activity in the developing chick heart

Net DNA synthesis continues throughout the embryonic development of chick ventricular tissue but the rate of DNA accumulation declines during the perinatal period. This slowing of DNA accumulation is paralleled by a decreased capacity of chick ventricular slices and of perfused whole hearts to incorporate ³H-thymidine into DNA. Synthesis of DNA by slices and whole hearts is completely inhibited by cytosine arabinoside (ara-C).At least two classes of DNA polymerase which are dependent upon exogenous DNA have been measured in the 100,000 g suppernatant fraction of chick ventricular homogenates. The predominant polymerase, active with a denatured DNA primer, exhibits a decline in activity which is correlated with the fall-off in DNA synthesis in ventricular tissue. The activity of a second DNA polymerase, active with a native DNA primer, remains constant throughout the developmental stages examined. The decrease in polymerase activity with a denatured DNA primer cannot be ascribed to soluble inhibitors of the polymerase or to detectable DNase activity in older myocardial tissue. Several characteristics of the crude enzyme have been examined, including primer and substrate dependence, glycerol and magnesium ion optima, and enzyme inhibition with N-ethylmaleimide (NEM) and 1-β-d-arabinofuranosylcytosine triphosphate (ara-CTP). Polymerase activity with denatured and native DNA primers is differentially susceptible to these reagents.     

Effect of polyamine limitation on DNA synthesis and development of mouse preimplantation embryos in vitro

In-vitro treatment of preimplantation mouse embryos with spermine and spermidine biosynthesis inhibitor, methylglyoxal-bis-(guanylhydrazone) (MGBG), arrested embryo development at the 8-cell or morula stage. In addition, the embryo DNA synthetic rate, as measured by [3H]thymidine incorporation, was strongly inhibited. The inhibition of blastocyst formation and DNA synthesis by MGBG was readily reversible by an exogenous supply of spermine and/or spermidine to the culture medium. DL-alpha-Methylornithine or DL-alpha-difluoromethylornithine (alpha-DFMO), inhibitors of putrescine biosynthesis, had no effect on embryos cultured for 1 or 2 days, but on the 3rd day embryo DNA synthesis was significantly depressed in the presence of alpha-DFMO. These observations suggest that, during early development of the preimplantation mouse embryo, spermine and spermidine are involved in regulation of embryo growth and DNA synthesis. They may also indicate a role of putrescine at a later stage of mouse embryo development.     

The role of calcium in DNA synthesis and development of mouse preimplantation embryos in vitro.

The effect of calcium upon embryonic growth was studied using cultured mouse preimplantation embryos. Both morphological development of the embryos and embryo DNA synthesis were shown to be dependent on the Ca2+ concentration in the medium in which the embryos were grown. Reduction of the calcium concentration below 10(-5) M completely blocked cell division and blastocyst formation in the cultured embryos, but only moderately inhibited embryo DNA synthesis. Trifluoperazine, a calmodulin antagonist, strongly inhibited the Ca(2+)-dependent DNA synthesis in the embryos. On the other hand, the drug only slightly affected the morphological development of the embryos. These results demonstrate that calcium independently affects two different aspects of the embryo development, i.e. DNA synthesis and cell division. It is suggested that the former effect is calmodulin-dependent, while the latter involves the calcium-dependence of metabolite transport through the cell membranes.     

Localized activity of Ca2+-stimulated ATPase and transcellular ionic currents during mesoderm induction in embryos ofLymnaea stagnalis (Mollusca)

Summary InLymnaea stagnalis, mesoderm induction occurs at the 24-cell stage, when the apical tip of the macromere 3D establishes a close contact with a number of micromeres. Via its tip, the macromere 3D is supposed to receive an inductive signal from the micromeres, resulting in the determination of the mesodermal stem cell 4d at the next division. In view of the possibility that transcellular ionic currents might somehow be involved, either in the processes that bring about this particular configuration of blastomeres or in the induction process itself, we mapped the electric field around the embryo during the 24-cell stage, using a vibrating probe. We detected a reversal of the current direction as compared to the uncleaved egg, whilst the polarity of the field along the animal-vegetal axis was maintained. We also mapped the localization of Ca²⁺-stimulated AT-Pase, an enzyme that drives the Ca²⁺-efflux from the cell. We found that this enzyme is localized exclusively along the cytoplasmic face of the apical plasma membrane of macromere 3D, and that its presence is restricted to the period from 110 to 135 min after the fifth cleavage, when there is close contact between macormere 3D and the micromeres. Since the localization of the Ca²⁺-stimulated ATPase coincides both in time and space with the induction of the mesoderm-mother cell, we suggest that localized calcium fluxes may play a role in this induction process.