Regulation of cell adhesion and collective cell migration by hindsight and its human homolog RREB1

Cell movements represent a major driving force in embryonic development, tissue repair, and tumor metastasis [1]. The migration of single cells has been well studied, predominantly in cell culture [2, 3]; however, in vivo, a greater variety of modes of cell movement occur, including the movements of cells in clusters, strands, sheets, and tubes, also known as collective cell migrations [4, 5]. In spite of the relevance of these types of movements in both normal and pathological conditions, the molecular mechanisms that control them remain predominantly unknown. Epithelial follicle cells of the Drosophila ovary undergo several dynamic morphological changes, providing a genetically tractable model [6]. We found that anterior follicle cells, including border cells, mutant for the gene hindsight (hnt) accumulated excess cell-cell adhesion molecules and failed to undergo their normal collective movements. In addition, HNT affected border cell cluster cohesion and motility via effects on the JNK and STAT pathways, respectively. Interestingly, reduction of expression of the mammalian homolog of HNT, RREB1, by siRNA inhibited collective cell migration in a scratch-wound healing assay of MCF10A mammary epithelial cells, suppressed surface activity, retarded cell spreading after plating, and led to the formation of immobile, tightly adherent cell colonies. We propose that HNT and RREB1 are essential to reduce cell-cell adhesion when epithelial cells within an interconnected group undergo dynamic changes in cell shape.     

Transformation of a grain legume (Lupinus angustifolius L.) via Agrobacterium tumefaciens-mediated gene transfer to shoot apices

Transgenic plants of Lupinus angustifolius L. (cvs. Unicrop and Merrit) were routinely generated using Agrobacterium-mediated gene transfer to shoot apices. The bar gene for resistance to phosphinothricin (PPT, the active ingredient of the herbicide Basta) was used as the selectable marker. After co-cultivation, the shoot apex explants were transferred onto a PPT-free regeneration medium and their tops were thoroughly wetted with PPT solution (2 mg/ml). The multiple axillary shoots developing from the shoot apices were excised onto a medium containing 20 mg/l PPT. The surviving shoots were transferred every second week onto fresh medium containing 20 mg/l PPT. At each transfer, the number of surviving shoots decreased, until it stabilized. Indeed, some of these chimeric shoots surviving the PPT selection, eventually produced new green healthier axillary shoots which could be transferred to soil. This whole process took from 5 to 9 months after co-cultivation. Average transformation frequencies of 2.8% for cv. Unicrop and of 0.4% for the commercial cultivar Merrit were achieved. Molecular analysis of T₀, T₁, and T₂ generations demonstrated stable integration of the foreign gene into the plant genome and expression of the integrated gene. Transformed plants of the T₁ and T₂ generations were resistant in glasshouse trials where the herbicide Basta (0.1 mg/ml) was sprayed onto whole plants. These results demonstrate that Agrobacterium-mediated gene transfer to preorganised meristematic tissue combined with axillary regeneration can form the basis of a routine transformation system for legume crop species which are difficult to regenerate from other explants.     

Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens

RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ～10 000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.     

Global scale variation in the salinity sensitivity of riverine macroinvertebrates: eastern Australia, France, Israel and South Africa

Salinity is a key abiotic property of inland waters; it has a major influence on biotic communities and is affected by many natural and anthropogenic processes. Salinity of inland waters tends to increase with aridity, and biota of inland waters may have evolved greater salt tolerance in more arid regions. Here we compare the sensitivity of stream macroinvertebrate species to salinity from a relatively wet region in France (Lorraine and Brittany) to that in three relatively arid regions eastern Australia (Victoria, Queensland and Tasmania), South Africa (south-east of the Eastern Cape Province) and Israel using the identical experimental method in all locations. The species whose salinity tolerance was tested, were somewhat more salt tolerant in eastern Australia and South Africa than France, with those in Israel being intermediate. However, by far the greatest source of variation in species sensitivity was between taxonomic groups (Order and Class) and not between the regions. We used a bayesian statistical model to estimate the species sensitivity distributions (SSDs) for salinity in eastern Australia and France adjusting for the assemblages of species in these regions. The assemblage in France was slightly more salinity sensitive than that in eastern Australia. We therefore suggest that regional salinity sensitivity is therefore likely to depend most on the taxonomic composition of respective macroinvertebrate assemblages. On this basis it would be possible to screen rivers globally for risk from salinisation.