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

Fluorescence Live Imaging of <i>Drosophila</i> Circadian Pacemaker Neurons

Live imaging of the molecular clockwork within the circadian pacemaker neurons offers the unique possibility to study complex interactions between the molecular clock and neuronal communication within individual neurons and throughout the entire circadian circuitry. Here we describe how to establish brain explants and dissociated neuron culture from Drosophila larvae, guidelines for time-lapse fluorescence microscopy, and the method of image analysis. This approach enables the long-term monitoring of fluorescence signals of circadian reporters at single-cell resolution and can be also applicable to analyze real-time expression of other fluorescent probes in Drosophila neurons.</p

Toward preclinical predictive drug testing for metabolism and hepatotoxicity by using in vitro models derived from human embryonic stem cells and human cell lines - a report on the Vitrocellomics EU-project.

Drug-induced liver injury is a common reason for drug attrition in late clinical phases, and even for post-launch withdrawals. As a consequence, there is a broad consensus in the pharmaceutical industry, and within regulatory authorities, that a significant improvement of the current in vitro test methodologies for accurate assessment and prediction of such adverse effects is needed. For this purpose, appropriate in vivo-like hepatic in vitro models are necessary, in addition to novel sources of human hepatocytes. In this report, we describe recent and ongoing research toward the use of human embryonic stem cell (hESC)-derived hepatic cells, in conjunction with new and improved test methods, for evaluating drug metabolism and hepatotoxicity. Recent progress on the directed differentiation of human embryonic stem cells to the functional hepatic phenotype is reported, as well as the development and adaptation of bioreactors and toxicity assay technologies for the testing of hepatic cells. The aim of achieving a testing platform for metabolism and hepatotoxicity assessment, based on hESC-derived hepatic cells, has advanced markedly in the last 2-3 years. However, great challenges still remain, before such new test systems could be routinely used by the industry. In particular, we give an overview of results from the Vitrocellomics project (EU Framework 6) and discuss these in relation to the current state-of-the-art and the remaining difficulties, with suggestions on how to proceed before such in vitro systems can be implemented in industrial discovery and development settings and in regulatory acceptance

Fluorescence circadian imaging reveals a PDF-dependent transcriptional regulation of the Drosophila molecular clock

Circadian locomotor behaviour is controlled by a pacemaker circuit composed of clock-containing neurons. To interrogate the mechanistic relationship between the molecular clockwork and network communication critical to the operation of the Drosophila circadian pacemaker circuit, we established new fluorescent circadian reporters that permit single-cell recording of transcriptional and post-transcriptional rhythms in brain explants and cultured neurons. Live-imaging experiments combined with pharmacological and genetic manipulations demonstrate that the neuropeptide pigment-dispersing factor (PDF) amplifies the molecular rhythms via time-of-day- and activity-dependent upregulation of transcription from E-box-containing clock gene promoters within key pacemaker neurons. The effect of PDF on clock gene transcription and the known role of PDF in enhancing PER/TIM stability occur via independent pathways downstream of the PDF receptor, the former through a cAMP-independent mechanism and the latter through a cAMP-PKA dependent mechanism. These results confirm and extend the mechanistic understanding of the role of PDF in controlling the synchrony of the pacemaker neurons. More broadly, our results establish the utility of the new live-imaging tools for the study of molecular-neural interactions important for the operation of the circadian pacemaker circuit