Automatic reconstruction of 3D neuron structures using a graph-augmented deformable model

Motivation: Digital reconstruction of 3D neuron structures is an important step toward reverse engineering the wiring and functions of a brain. However, despite a number of existing studies, this task is still challenging, especially when a 3D microscopic image has low single-to-noise ratio and discontinued segments of neurite patterns

Gene Activation Using FLP Recombinase in C. elegans

The FLP enzyme catalyzes recombination between specific target sequences in DNA. Here we use FLP to temporally and spatially control gene expression in the nematode C. elegans. Transcription is blocked by the presence of an “off cassette” between the promoter and the coding region of the desired product. The “off cassette” is composed of a transcriptional terminator flanked by FLP recognition targets (FRT). This sequence can be excised by FLP recombinase to bring together the promoter and the coding region. We have introduced two fluorescent reporters into the system: a red reporter for promoter activity prior to FLP expression and a green reporter for expression of the gene of interest after FLP expression. The constructs are designed using the multisite Gateway system, so that promoters and coding regions can be quickly mixed and matched. We demonstrate that heat-shock-driven FLP recombinase adds temporal control on top of tissue specific expression provided by the transgene promoter. In addition, the temporal switch is permanent, rather than acute, as is usually the case for heat-shock driven transgenes. Finally, FLP expression can be driven by a tissue specific promoter to provide expression in a subset of cells that can only be addressed as the intersection of two available promoters. As a test of the system, we have driven the light chain of tetanus toxin, a protease that cleaves the synaptic vesicle protein synaptobrevin. We show that we can use this to inactivate synaptic transmission in all neurons or a subset of neurons in a FLP-dependent manner

Genome-Wide Analysis of Light- and Temperature-Entrained Circadian Transcripts in Caenorhabditis elegans

Transcriptional profiling experiments identify light- and temperature-entrained circadian transcripts in C. elegans

Targeted cell killing by reconstituted caspases

We have developed a two-component system involving reconstituted caspase (recCaspase) for selective and/or conditional ablation of targeted cells. Caspases, the executioners of programmed cell death, are normally synthesized as inactive zymogens and are activated by proteolytic processing of their subunits. We show here, using two different caspases, Caenorhabditis elegans CED-3 and human Caspase-3, that coexpression of the subunits generates constitutively active caspase activity that leads to cell death. This recCaspase activity, however, occurred only when the subunits associated through binding of linked antiparallel leucine-zipper domains. We exploited the dual-component nature of recCaspases by expressing the individual subunits from combinations of promoters either to target selectively the subset of cells for apoptosis or induce cell death in specific cells at specific times during development. The high degree of target specificity and tight regulation of induction of recCaspase would be advantageous in creating animal models that are ablated for specific cells and in other targeted cell killings