Roles of β-catenin in somitogenesis in rat embryos

Summary We studied the roles of β-catenin in somitogenesis using immunostaining and antisense experiments in rat embryos. High levels of β-catenin appeared transiently in the developing rat somites. Initially, β-catenin accumulation was observed in the core cells of presomitic cell aggregates and then in the lumen of epithelial vesicles. Subsequently, it was confined to the dermomyotomes and their lumen and then the myotomes. High levels of cyclin D1 were observed in the core cells, in the lumen of epithelial vesicles, in myotomes, and in mesenchymal sclerotomes. When embryos were cultured in medium supplemented with β-catenin antisense oligodeoxynucleotide (ODN), the accumulation of β-catenin, but not of cyclin D1, in the nascent somites and dermomyotomes was suppressed, while the number of somites was the same as that observed in control embryos. The number of myosin-positive somites and the amount of myosin per somite in embryos treated with the antisense ODN were lower than those in controls. These results suggested that β-catenin promotes development of myotomal cells during somitogenesis. The function of β-catenin in the development of myotomes may not be correlated to cyclin D1.     

Alterations in glycosaminoglycan metabolism in β-aminopropionitrile-treated chick embryos

1. Na(2) (35)SO(4), [1-(14)C]glucosamine and [1-(14)C]acetate were used as precursors of the sulphated glycosaminoglycans to study the biochemical effect of beta-aminopropionitrile in chick embryos. The incorporation of all three precursors was decreased in the treated embryos between days 7 and 10 of embryonic development. No inhibition of incorporation of these precursors occurred between days 16 and 20 of embryonic development at the dosages of beta-aminopropionitrile used. 2. beta-Aminopropionitrile treatment also decreased the amount of N-acetylhexosamines in the chick embryo and decreased the percentage of the hexosamine esterified by nucleotides. Respiration was decreased by homogenates prepared from treated embryos. Likewise, UDP-xylosyl- and UDP-galactosyl-transferase activities were decreased in treated embryos and cartilage from embryos and growing chicks. 3. The data suggest that beta-aminopropionitrile, in addition to the known lathyrogenic activity, either is or gives rise to a potent metabolic poison that interferes with basic cellular metabolism. The results are consistent with a decreased rate of ATP generation as an explanation for the decrease in glycosaminoglycan synthesis.     

Sugar sensing and α-amylase gene repression in rice embryos

We used a transient expression system to study the mechanism by which carbohydrates repress a rice (Oryza sativa L.) α-amylase (EC 3.2.1.1) gene. Exogenously fed metabolizable carbohydrates are able to elicit repression of the α-amylase gene RAmy3D in the rice embryo, and our results indicate that repression is also triggered efficiently by endogenous carbohydrates. Glucose analogs that are taken up by plant cells but not phosphorylated by hexokinase are unable to repress the α-amylase gene studied, while 2-deoxyglucose, which is phosphorylable but not further metabolized, down-regulates RAmy3D promoter activity, indicating a role for hexokinase in the sugar-sensing mechanism triggering repression of the RAmy3D gene. We tested two different hexokinase inhibitors, mannoheptulose and glucosamine, but only the latter was able to relieve RAmy3D promoter activity from repression by endogenous carbohydrates. This correlates with the higher ability of glucosamine to inhibit the activity of rice hexokinases in vitro. The glucosamine-mediated relief of RAmy3D promoter activity from repression by endogenous carbohydrates does not correlate with a reduced rate of carbohydrate utilization. Received: 22 April 1997 / Accepted: 9 September 1997     

Integrin βν-mediated phagocytosis of apoptotic cells in Drosophila embryos

To identify molecules that play roles in the clearance of apoptotic cells by Drosophila phagocytes, we examined a series of monoclonal antibodies raised against larval hemocytes for effects on phagocytosis in vitro. One antibody that inhibited phagocytosis recognized terribly reduced optic lobes (Trol), a core protein of the perlecan-type proteoglycan, and the level of phagocytosis in embryos of a Trol-lacking fly line was lower than in a control line. The treatment of a hemocyte cell line with a recombinant Trol protein containing the amino acid sequence RGD augmented the phosphorylation of focal adhesion kinase, a hallmark of integrin activation. A loss of integrin βν, one of the two β subunits of Drosophila integrin, brought about a reduction in the level of apoptotic cell clearance in embryos. The presence of integrin βν at the surface of embryonic hemocytes was confirmed, and forced expression of integrin βν in hemocytes of an integrin βν-lacking fly line recovered the defective phenotype of phagocytosis. Finally, the level of phagocytosis in a fly line that lacks both integrin βν and Draper, another receptor required for the phagocytosis of apoptotic cells, was lower than that in a fly line lacking either protein. We suggest that integrin βν serves as a phagocytosis receptor responsible for the clearance of apoptotic cells in Drosophila, independent of Draper.     

Lack of centrioles and primary cilia in STIL−/− mouse embryos

Although most animal cells contain centrosomes, consisting of a pair of centrioles, their precise contribution to cell division and embryonic development is unclear. Genetic ablation of STIL, an essential component of the centriole replication machinery in mammalian cells, causes embryonic lethality in mice around mid gestation associated with defective Hedgehog signaling. Here, we describe, by focused ion beam scanning electron microscopy, that STIL^−/− mouse embryos do not contain centrioles or primary cilia, suggesting that these organelles are not essential for mammalian development until mid gestation. We further show that the lack of primary cilia explains the absence of Hedgehog signaling in STIL^−/− cells. Exogenous re-expression of STIL or STIL microcephaly mutants compatible with human survival, induced non-templated, de novo generation of centrioles in STIL^−/− cells. Thus, while the abscence of centrioles is compatible with mammalian gastrulation, lack of centrioles and primary cilia impairs Hedgehog signaling and further embryonic development.     

The expression of β-glucuronidase during preimplantation development of mouse embryos

β-Glucuronidase activity was measured in mouse embryos during the preimplantation period of development by using a microfluorometric assay. A 100-fold increase in activity was observed between 57 (8-cell stage) and 84 hr (morulae) of development. Activity changes between 30 and 60 hr were also significant. Genetic variants of β-glucuronidase occur between the strains of mice $\text{C57BL}\text{6J}$ and $\text{C3H}\text{HeJ}$ which differ in levels of activity and heat denaturation kinetics. Activity changes and heat denaturation kinetics of β-glucuronidase in $\text{C57BL}\text{6}\text{,}\text{C3H}\text{HeJ}$ and F₁ hybrid embryos were compared, and it was demonstrated that paternal genes were expressed during the 100-fold increase in activity and that embryonic genes may be functioning between 30 and 60 hr of development.     

The evolution of early animal embryos: conservation or divergence?

There is a remarkable similarity in the appearance of groups of animal species during periods of their embryonic development. This classic observation has long been viewed as an emphatic realization of the principle of common descent. Despite the importance of embryonic conservation as a unifying concept, models seeking to predict and explain different patterns of conservation have remained in contention. Here, we focus on early embryonic development and discuss several lines of evidence, from recent molecular data, through developmental networks to life-history strategies, that indicate that early animal embryos are not highly conserved. Bringing this evidence together, we argue that the nature of early development often reflects adaptation to diverse ecological niches. Finally, we synthesize old and new ideas to propose a model that accounts for the evolutionary process by which embryos have come to be conserved.     

Control of cleavage cycles in Drosophila embryos by frühstart

Early metazoan development consists of cleavage stages characterized by rapid cell cycles that successively divide the fertilized egg. The cell cycle oscillator pauses when the ratio of DNA and cytoplasm (N/C) reaches a threshold characteristic for the species. This pause requires maternal factors as well as zygotic expression of as yet unknown genes. Here we isolate the zygotic gene frühstart of Drosophila and show that it is involved in pausing the cleavage cell cycle. frs is expressed immediately after the last cleavage division. It plays a role in generating a uniform pause and it can inhibit cleavage divisions when precociously expressed. Furthermore, the expression of frs is delayed in haploid embryos and requires activity of the maternal checkpoint gene grapes. We propose that zygotic frs expression is involved in linking the N/C and the pause of cleavage cycle.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

Can embryo metabolism be used for selecting bovine embryos before transfer?

The quality of in vitro-produced bovine embryos remains variable. The selection of these embryos based only on their morphology does not allow for acceptable gestational rates to be obtained. The use of metabolic markers to select viable embryos before transfer would be of valuable help, both economically and as a research tool. The ideal marker should meet several conditions: it should be able to be evaluated 1) in a totally non-invasive manner, 2) on individual embryos (which necessitates very sensitive techniques), 3) very rapidly (so that it is compatible with the immediate transfer of fresh embryos), and 4) in order to allow viable embryos to be separated from those that are not viable, whatever the production system used. In practice, such a marker does not exist, but certain methods of metabolic evaluation resemble it. The development of a metabolic marker is confronted by the metabolic characteristics of the embryo, notably the evolution of the metabolism during the development of the embryo and its adaptation to the changes in the environment.     

A “micromere model” of cell-cell interactions in sea urchin early embryos

It has been shown that isolation of sea urchin blastomeres before the post-division adhesion leads mainly to the formation of equal blastomeres at the stage of 4th cleavage division, whereas isolation after adhesion results in the formation of micromeres simultaneous with that in intact embryos. Similar results were obtained in five sea urchin species. It has been concluded that there exists a critical point in the cleavage process, when blastomeres exchange information that determines the further cleavage pattern. It has been shown with this “micromere model” that serotonin and its analogs influence the cleavage pattern of half-embryos. These data have served as a basis for the hypothesis of “protosynapse,” a bilaterally symmetric structure in which the blastomeres are not only source and target of the signal but also a passive obstacle to leakage of the signal substance from the interblastomere cleft to the milieu. Such a structure may also specify the primary asymmetry of the blastomeres. The micromere model may be useful in specific pharmacological screening.     

Survival after transfer of “sexed” mouse embryos exposed to H-Y antisera

Immunological means were used to determine the sex of mouse embryos prior to transfer to pseudopregnant recipients. Antisera to histocompatibility-Y (H-Y) antigen were prepared in adult C57BL/6 female mice by repeated intraperitoneal injections of spleen cells from males of the same strain. Eight-to 16-cell embryos were cultured in BMOC-3 alone or BMOC-3 without bovine serum albumin to which one of the following had been added: H-Y antiserum and normal guinea pig serum (NGPS), NGPS alone, normal mouse serum alone or normal mouse serum and NGPS. After 24 hr of culture, embryos were classified as either affected or unaffected. An embryo was classified as affected if degeneration of the embryo or breakdown of one or more cells was observed. A total of 1000 embryos were cultured in BMOC-3 with H-Y antiserum and NGPS (treated embryos). Two hundred and fifty embryos were cultured in each of the other four media (control embryos). Eighty-seven (9%) of the control embryos and 479 (48%) of the treated embryos were classified as affected after culture. Unaffected embryos, approximately 12 each, were transferred to pseudopregnant recipients. One-hundred forty control embryos (17%) survived to term with 67 females (48%) and 73 males (52%) born. Fifty-eight treated embryos (14%) survived to term, producing 50 females (86%) and 8 males (14%). Percentage of females from embryos cultured in antiserum was greater than for embryos cultured in any other media (P<0.001). These results demonstrate that detection of H-Y antigen on preimplantation embryos may be a useful and effective method of determining sex of an embryo prior to transfer.     

Induction of petal-bearing embryos from root-derived callus of Oncidium ‘Gower Ramsey’

A repeatable in vitro culture method was established and it could induce a series of abnormal embryos by which their scale leaves were substituted by petals. These petal-bearing embryos were derived from long-term root calli of an orchid cultivar, Oncidium ??Gower Ramsey??. The calli were induced and subcultured on a modified 1/2MS medium supplemented with five combinations of TDZ and dicamba. When 1-year-old callus, which induced and subcultured at 3?mg/l TDZ and 5?mg/l dicamba (line 13 callus), was transferred onto 1/2MS medium supplemented with 0.1?ml/l NAA and 3?mg/l TDZ, it gave the highest number of petal-bearing embryos. However, line 13 root explants gave one of the lowest percentage of callus formation (12.5%) and the number of somatic embryos per callus was also one of the lowest (along with lines 10, 11 and 12). The efficiency of embryogenesis decreased with age of callus and the significant decrease was started from the third year of culturing. Flowering characteristics of plantlets from normal embryos and petal-bearing embryos were both evaluated after 3?years of culture in the greenhouse. Parameters including length of the longest inflorescence, numbers of flowers per plant, length of flowers and width of flowers were all not significantly different between abnormal embryo-derived plants and normal embryo-derived plants.