Drosophilin C

The genome is organized into topologically associating domains (TADs) delimited by boundaries that isolate interactions between domains. In Drosophila, the mechanisms underlying TAD formation and boundaries are still under investigation. The application of the in-nucleus Hi-C method described here helped to dissect the function of architectural protein (AP)-binding sites at TAD boundaries isolating the Notch gene. Genetic modification of domain boundaries that cause loss of APs results in TAD fusion, transcriptional defects, and long-range topological alterations. These results provided evidence demonstrating the contribution of genetic elements to domain boundary formation and gene expression control in Drosophila. Here, the in-nucleus Hi-C method has been described in detail, which provides important checkpoints to assess the quality of the experiment along with the protocol. Also shown are the required numbers of sequencing reads and valid Hi-C pairs to analyze genomic interactions at different genomic scales. CRISPR/Cas9-mediated genetic editing of regulatory elements and high-resolution profiling of genomic interactions using this in-nucleus Hi-C protocol could be a powerful combination for the investigation of the structural function of genetic elements.

Many organisms-from bacteria to nematodes to insect larvae-navigate their environments by biasing random movements. In these organisms, navigation in isotropic environments can be characterized as an essentially diffusive and undirected process. In stimulus gradients, movement decisions are biased to drive directed navigation toward favorable environments. How does directed navigation in a gradient modulate random exploration either parallel or orthogonal to the gradient? Here, we introduce methods originally used for analyzing protein folding trajectories to study the trajectories of the nematode Caenorhabditis elegans and the Drosophila larva in isotropic environments, as well as in thermal and chemical gradients. We find that the statistics of random exploration in any direction are little affected by directed movement along a stimulus gradient. A key constraint on the behavioral strategies of these organisms appears to be the preservation of their capacity to continuously explore their environments in all directions even while moving toward favorable conditions.

The full functionality of the brain is determined by its molecular, cellular and circuit structure. Modern neuroscience now prioritizes the mapping of whole brain connectomes by detecting all direct neuron to neuron synaptic connections, a feat first accomplished for C. elegans, a full reconstruction of a 302-neuron nervous system. Efforts at Janelia Research Campus will soon reconstruct the whole brain connectomes of a larval and an adult Drosophila. These connectomes will provide a framework for incorporating detailed neural circuit information that Drosophila neuroscientists have gathered over decades. But when viewed in the context of a whole brain, it becomes difficult to isolate the contributions of distinct circuits, whether sensory systems or higher brain regions. The complete wiring diagram tells us that sensory information is not only processed in separate channels, but that even the earliest sensory layers are strongly synaptically interconnected. In the higher brain, long-range projections densely interconnect major brain regions and convergence centers that integrate input from different sensory systems. Furthermore, we also need to understand the impact of neuronal communication beyond direct synaptic modulation. Nevertheless, all of this can be pursued with Drosophila, combining connectomics with a diverse array of genetic tools and behavioral paradigms that provide effective approaches to entire brain function.