The developing nervous system consists of a variety of cell types. Transgenic animals expressing reporter genes in specific classes of neuronal cells are powerful tools for the study of neuronal network formation. We generated a wide variety of transgenic zebrafish that expressed reporter genes in specific classes of neurons or neuronal progenitors. These include lines in which neurons of specific neurotransmitter phenotypes expressed fluorescent proteins or Gal4, and lines in which specific subsets of the dorsal progenitor domain in the spinal cord expressed fluorescent proteins. Using these, we examined domain organization in the developing dorsal spinal cord, and found that there are six progenitor domains in zebrafish, which is similar to the domain organization in mice. We also systematically characterized neurotransmitter properties of the neurons that are produced from each domain. Given that reporter gene expressions occurs in a wide area of the nervous system in the lines generated, these transgenic fish should serve as powerful tools for the investigation of not only the neurons in the dorsal spinal cord but also neuronal structures and functions in many other regions of the nervous system.

We study transitivity properties of edge weights in complex networks. We show that enforcing transitivity leads to a transitivity inequality which is equivalent to ultra-metric inequality. This can be used to define transitive closure on weighted undirected graphs, which can be computed using a modified Floyd-Warshall algorithm. These new concepts are extended to dissimilarity graphs and triangle inequalities. From this, we extend the clique concept from unweighted graph to weighted graph. We outline several applications and present results of detecting protein functional modules in a protein interaction network.

Translation is the fundamental biological process converting mRNA information into proteins. Single molecule imaging in live cells has illuminated the dynamics of RNA transcription; however, it is not yet applicable to translation. Here we report Single molecule Imaging of NAscent PeptideS (SINAPS) to assess translation in live cells. The approach provides direct readout of initiation, elongation, and location of translation. We show that mRNAs coding for endoplasmic reticulum (ER) proteins are translated when they encounter the ER membrane. Single molecule fluorescence recovery after photobleaching provides direct measurement of elongation speed (5 AA/s). In primary neurons mRNAs are translated in proximal dendrites but repressed in distal dendrites and display “bursting” translation. This technology provides a tool to address the spatiotemporal translation mechanism of single mRNAs in living cells.

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

Drosophila larval locomotion, which entails rhythmic body contractions, is controlled by sensory feedback from proprioceptors. The molecular mechanisms mediating this feedback are little understood. By using genetic knock-in and immunostaining, we found that the Drosophila melanogaster transmembrane channel-like (tmc) gene is expressed in the larval class I and class II dendritic arborization (da) neurons and bipolar dendrite (bd) neurons, both of which are known to provide sensory feedback for larval locomotion. Larvae with knockdown or loss of tmc function displayed reduced crawling speeds, increased head cast frequencies, and enhanced backward locomotion. Expressing Drosophila TMC or mammalian TMC1 and/or TMC2 in the tmc-positive neurons rescued these mutant phenotypes. Bending of the larval body activated the tmc-positive neurons, and in tmc mutants this bending response was impaired. This implicates TMC's roles in Drosophila proprioception and the sensory control of larval locomotion. It also provides evidence for a functional conservation between Drosophila and mammalian TMCs.

Besides 19,008 possible ectodomains, Drosophila Dscam contains two alternative transmembrane/juxtamembrane segments, respectively, derived from exon 17.1 and exon 17.2. We wondered whether specific Dscam isoforms mediate formation and segregation of axonal branches in the Drosophila mushroom bodies (MBs). Removal of various subsets of the 12 exon 4s does not affect MB neuronal morphogenesis, while expression of a Dscam transgene only partially rescues Dscam mutant phenotypes. Interestingly, differential rescuing effects are observed between two Dscam transgenes that each possesses one of the two possible exon 17s. Axon bifurcation/segregation abnormalities are better rescued by the exon 17.2-containing transgene, but coexpression of both transgenes is required for rescuing mutant viability. Meanwhile, exon 17.1 targets ectopically expressed Dscam-GFP to dendrites while Dscam[exon 17.2]-GFP is enriched in axons; only Dscam[exon 17.2] affects MB axons. These results suggest that exon 17.1 is minimally involved in axonal morphogenesis and that morphogenesis of MB axons probably involves multiple distinct exon 17.2-containing Dscam isoforms.

Vimentin intermediate filaments (VIFs) form complex, tight-packed networks; due to this density, traditional ensemble labeling and imaging approaches cannot accurately discern single filament behavior. To address this, we introduce a sparse vimentin-SunTag labeling strategy to unambiguously visualize individual filament dynamics. This technique confirmed known long-range dynein and kinesin transport of peripheral VIFs and uncovered extensive bidirectional VIF motion within the perinuclear vimentin network, a region we had thought too densely bundled to permit such motility. To examine the nanoscale organization of perinuclear vimentin, we acquired high-resolution electron microscopy volumes of a vitreously frozen cell and reconstructed VIFs and microtubules within a 50 um3 window. Of 583 VIFs identified, most were integrated into long, semi-coherent bundles that fluctuated in width and filament packing density. Unexpectedly, VIFs displayed minimal local co-alignment with microtubules, save for sporadic cross-over sites that we predict facilitate cytoskeletal crosstalk. Overall, this work demonstrates single VIF dynamics and organization in the cellular milieu for the first time.

At the center of the secretory pathway, the Golgi complex ensures correct processing and sorting of cargos toward their final destination. Cargos are diverse in topology, function and destination. A remarkable feature of the Golgi complex is its ability to sort and process these diverse cargos destined for secretion, the cell surface, the lysosome, or retained within the secretory pathway. Just as these cargos are diverse so also are their sorting requirements and thus, their trafficking route. There is no one-size-fits-all sorting scheme in the Golgi. We propose a coexistence of models to reconcile these diverse needs. We review examples of differential sorting mediated by proteins and lipids. Additionally, we highlight recent technological developments that have potential to uncover new modes of transport.

The stability of elements of three different dispersed repeated gene families in the genome of Drosophila tissue culture cells has been examined. Different amounts of sequences homologous to elements of 412, copia and 297 dispersed repeated gene families are found in the genomes of D. melanogaster embryonic and tissue culture cells. In general the amount of these sequences is increased in the cell lines. The additional sequences homologous to 412, copia and 297 occur as intact elements and are dispersed to new sites in the cell culture genome. It appears that these elements can insert at many alternative sites. We also describe a DNA sequence arrangement found in the D. melanogaster embryo genome which appears to result from a transposition of an element of the copia dispersed repeated gene family into a new chromosomal site. The mechanism of insertion of this copia element is precise to within 90 bp and may involve a region of weak sequence homology between the site of insertion and the direct terminal repeats of the copia element.

Our understanding of the mechanisms of neural circuit assembly is far from complete. Identification of wiring molecules with novel mechanisms of action will provide insights into how complex and heterogeneous neural circuits assemble during development. In the olfactory system, 50 classes of olfactory receptor neurons (ORNs) make precise synaptic connections with 50 classes of partner projection neurons (PNs). Here, we performed an RNA interference screen for cell surface molecules and identified the leucine-rich repeat-containing transmembrane protein known as Fish-lips (Fili) as a novel wiring molecule in the assembly of the olfactory circuit. Fili contributes to the precise axon and dendrite targeting of a small subset of ORN and PN classes, respectively. Cell-type-specific expression and genetic analyses suggest that Fili sends a transsynaptic repulsive signal to neurites of nonpartner classes that prevents their targeting to inappropriate glomeruli in the antennal lobe.