Functional studies of signaling pathways in peri-implantation development of the mouse embryo by RNAi

Background

Studies of gene function in the mouse have relied mainly on gene targeting via homologous recombination. However, this approach is difficult to apply in specific windows of time, and to simultaneously knock-down multiple genes. Here we report an efficient method for dsRNA-mediated gene silencing in late cleavage-stage mouse embryos that permits examination of phenotypes at post-implantation stages.

Results

We show that introduction of Bmp4 dsRNA into intact blastocysts by electroporation recapitulates the genetic Bmp4 null phenotype at gastrulation. It also reveals a novel role for Bmp4 in the regulation the anterior visceral endoderm specific gene expression and its positioning. We also show that RNAi can be used to simultaneously target several genes. When applied to the three murine isoforms of Dishevelled, it leads to earlier defects than previously observed in double knock-outs. These include severe delays in post-implantation development and defects in the anterior midline and neural folds at headfold stages.

Conclusion

Our results indicate that the BMP4 signalling pathway contributes to the development of the anterior visceral endoderm, and reveal an early functional redundancy between the products of the murine Dishevelled genes. The proposed approach constitutes a powerful tool to screen the functions of genes that govern the development of the mouse embryo.     

Akt participation in the Wnt signaling pathway through Dishevelled

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.     

Behavioural and physiological consequences of acute social defeat in growing gilts: effects of the social environment

Endocrine, behavioural and immunologic processes, together with body growth, were evaluated in gilts that were defeated at 10 weeks of age in resident-intruder tests. Immediately after defeat, gilts were either separated from or reunited with a familiar conspecific (litter-mate; always a barrow). Gilts were assigned to one of four treatments: (a) DI: defeat, followed by isolation (separation from original litter-mate; n=8); (b) I: no defeat, isolation (control group; n=9); (c) DP; defeat, followed by pair-housing (reunion with original litter-mate; n=8); and (d) P: no defeat, pair-housing (control group; n=8). The following general conclusions were derived: (1) social defeat caused pronounced short-term elevations in hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal medullary activities, and of prolactin levels. Moreover, as soon as 1h after defeat, percentages of blood lymphocytes and neutrophilic granulocytes were, respectively, decreased and increased; (2) social defeat had some long-lasting influence on behaviour and physiology, but isolation predominantly determined responses in the longer term. Defeat, as well as isolation, resulted in increased cardiovascular activities compared to P controls, as observed in a novel object test (NOT: +7 days) and an aversion test (AVT: +14 days). Moreover, defeated as well as isolated gilts did not habituate to a repeated novel environment test (NET: -7, +2 and +7 days) in terms of frequencies of vocalising, whereas P controls did. Isolation, through the separation from any other pig, was responsible for the other observed long-term characteristics, which developed progressively. Isolated gilts showed high mobilities and high cortisol responses in the repeated NET (+7 days), not being habituated. This contrasted the reactions of pair-housed gilts, which were much reduced. In addition to their high cardiovascular activities in the NOT and the AVT, isolated gilts also displayed higher heart rates in the repeated NET and during human presence following the NOT, compared to pair-housed gilts. Finally, isolated gilts were more inhibited to approach a novel object (in the NOT) than pair-housed pigs; and (3) stress responses of defeated gilts were modulated by the subsequent social environment. Stimulation of the HPA-axis (plasma- and salivary cortisol) was prolonged in those defeated gilts which were isolated (observed in the first hour). Changes in leucocyte subsets were still observed after 3 days in DI, but were 'normalised' within 1 day in DP gilts. Two days after defeat, habituation to the repeated NET in terms of mobility and salivary cortisol responses occurred in control and DP gilts, but not in DI gilts. We argue that these effects of the social environment shortly after defeat were related to a stress-reducing effect of a stable social relationship, i.e. social support.     

Navigation and thermophotovoltaic activity by hornets at different light conditions: the influence of UVB blockers

The aim of the present investigation was two-fold: a) to observe the homing of the Oriental hornet, Vespa orientalis (Hymenoptera, Vespinae) from different distances; and b) to study the photothermoelectric activity of hornet cuticle obtained from the subjects of goal (a) and kept frozen for a number of days prior to its testing. In both the above mentioned phases of the investigation, an attempt was made to assess how the covering of the hornets' cuticle with Ultra Violet B (UVB) blockers affects their activity as compared to the control. Flying hornets were observed to return to the nest from distances of up to 7 km, once they had learned the way back. However, covering of the cuticle with UVB blockers increases the percentage of 'non-returners' to nearly 100%. Covering the cuticle completely or partly with a number of UVB blockers (except for Sisley) proves lethal for the hornets within 24 hours. A statistical model on homing is proposed of the effect of range, of covering with UVB blockers and covering ocelli with Tippex. In the wing of the hornet there is increase in the electric current with rise in the temperature and decrease in the current upon drop of the temperature, but light has no effect on this alar (wing) current. Contrariwise, the body cuticle of the hornet responds to both temperature and illumination in terms of its electric current. Coating of the cuticle with UVB blockers causes in the wing (under all conditions of illumination) and in the cuticle (only in the dark) a moderation in the amplitude of the photothermoelectric current.     

Characterization of aggregation and protein expression of bovine corneal endothelial cells as microcarrier cultures in a rotating-wall vessel

Rotating-wall vessels are beneficial to tissue engineering in that the reconstituted tissue formed in these low-shear bioreactors undergoes extensive three-dimensional growth and differentiation. In the present study, bovine corneal endothelial (BCE) cells were grown in a high-aspect rotating-wall vessel (HARV) attached to collagen-coated Cytodex-3 beads as a representative monolayer culture to investigate factors during HARV cultivation which affect three-dimensional growth and protein expression. A collagen type I substratum in T-flask control cultures increased cell density of BCE cells at confluence by 40% and altered the expression of select proteins (43, 50 and 210 kDa). The low-shear environment in the HARV facilitated cell bridging between microcarrier beads to form aggregates containing upwards of 23 beads each, but it did not promote multilayer growth. A kinetic model of microcarrier aggregation was developed which indicates that the rate of aggregation between a single bead and an aggregate was nearly 10 times faster than between two aggregate and 60 times faster than between two single beads. These differences reflect changes in collision frequency and cell bridge formation. HARV cultivation altered the expression of cellular proteins (43 and 70 kDa) and matrix proteins (50, 73, 89 and 210 kDa) relative to controls perhaps due to hypoxia, fluid flow or distortion of cell shape. In addition to the insight that this work has provided into rotating-wall vessels, it could be useful in modeling aggregation in other cell systems, propagating human corneal endothelial cells for eye surgery and examining the response of endothelial cells to reduced shear.     

Ex Uno Plures: Clonal Reinforcement Drives Evolution of a Simple Microbial Community

A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community''s underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103''s phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an “engine” generating diversity by creating new metabolic niches, but not the occupants themselves.