Identification of Neural Outgrowth Genes using Genome-Wide RNAi

While genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system

Expansion and Harvesting of hMSC-TERT

The expansion of human mesenchymal stem cells as suspension culture by means of spinner flasks and microcarriers, compared to the cultivation in tissue culture flasks, offers the advantage of reducing the requirements of large incubator capacities as well as reducing the handling effort during cultivation and harvesting. Nonporous microcarriers are preferable when the cells need to be kept in viable condition for further applications like tissue engineering or cell therapy. In this study, the qualification of Biosilon, Cytodex 1, Cytodex 3, RapidCell and P102-L for expansion of hMSC-TERT with an associated harvesting process using either trypsin, accutase, collagenase or a trypsin-accutase mixture was investigated. A subsequent adipogenic differentiation of harvested hMSC-TERT was performed in order to observe possible negative effects on their (adipogenic) differentiation potential as a result of the cultivation and harvesting method. The cultivated cells showed an average growth rate of 0.52 d-1. The cells cultivated on Biosilon, RapidCell and P102-L were harvested succesfully achieving high cell yield and vitalities near 100%. This was not the case for cells on Cytodex 1 and Cytodex 3. The trypsin-accutase mix was most effective. After spinner expansion and harvesting the cells were successfully differentiated to adipocytes

Automatic Robust Neurite Detection and Morphological Analysis of Neuronal Cell Cultures in High-content Screening

Cell-based high content screening (HCS) is becoming an important and increasingly favored approach in therapeutic drug discovery and functional genomics. In HCS, changes in cellular morphology and biomarker distributions provide an information-rich profile of cellular responses to experimental treatments such as small molecules or gene knockdown probes. One obstacle that currently exists with such cell-based assays is the availability of image processing algorithms that are capable of reliably and automatically analyzing large HCS image sets. HCS images of primary neuronal cell cultures are particularly challenging to analyze due to complex cellular morphology. Here we present a robust method for quantifying and statistically analyzing the morphology of neuronal cells in HCS images. The major advantages of our method over existing software lie in its capability to correct non-uniform illumination using the contrast-limited adaptive histogram equalization method; segment neuromeres using Gabor-wavelet texture analysis; and detect faint neurites by a novel phase-based neurite extraction algorithm that is invariant to changes in illumination and contrast and can accurately localize neurites. Our method was successfully applied to analyze a large HCS image set generated in a morphology screen for polyglutaminemediated neuronal toxicity using primary neuronal cell cultures derived from embryos of a Drosophila Huntington’s Disease (HD) model.National Institutes of Health (U.S.) (Grant

The discovery, distribution, and evolution of viruses associated with drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens, but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host-virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research

Characterization of early steps in muscle morphogenesis in a Drosophila primary culture system

Myogenesis in Drosophila embryos requires fusion between Founder cells (FCs) and Fusion Competent myoblasts (FCMs) to form multinucleate myotubes. Myoblast fusion is well characterized in embryos, and many factors required for this process have been identified; however, a number of questions pertaining to the mechanisms of fusion remain and are challenging to answer in the embryo. We have developed a modified primary cell culture protocol to address these questions in vitro. Using this system, we determined the optimal time for examining fusion in culture and confirmed that known fusion proteins are expressed and localized as in embryos. Importantly, we disrupted the actin and microtubule networks with the drugs latrunculin B and nocodazole, respectively, confirming that actin is required for myoblast fusion and showing for the first time that microtubules are also required for this process in Drosophila. Finally, we show that myotubes in culture adopt and maintain specific muscle identities