Growth cones advance by forward translocation of the C-domain and axonal framework <i>in</i><i>vivo</i>.

<p>(<b>A</b>) A 3D reconstruction of late stage 16 embryo expressing the membrane marker myr-tdTomato in the nervous system via <i>elav-Gal4</i>. After the intersegmental axon of the aCC neuron passes the point where the RP2 axon forms a synapse on muscle 2, it is in a region free of other axons and the cell bodies of surrounding sensory neurons. The box indicates the region of the aCC motor axon that was used for 3D analysis of mitochondrion advance. (<b>B</b> - <b>D</b>) Time-lapse series of an elongating <i>Drosophila</i> aCC motor neuron in stage 16 embryo of the genotype +/elav-Gal4;;<i>UAS-mtGFP</i>, <i>dmiro</i>^B682/ <i>IVS-10XUAS-myr-tdTom</i>, shown at 2 min intervals. (<b>B</b>) myr-tdTomato (red in <b>D</b>) labels neuronal plasma membranes. (<b>C</b>) mitoGFP (green in <b>D</b>) labels mitochondria. The arrow shows a mitochondrion in growth cone. In the last half of the series a mitochondrion docks in the distal axon (triangle in <b>B</b>) and advances. (<b>E</b>) Average velocity of docked mitochondria in the growth cone, defined as the last five µm of the axon, and in binned regions along the distal axon. Because the RP2 axon is fasciculated with the aCC axon (<b>A</b>), only mitochondria in the last 25 µm of the aCC axon were analyzed. Error bars show the 95% confidence intervals. The number at the base of the bar is the number of docked mitochondria that were analyzed. Scale bars = 10 µm.</p

Consensual Uncertainty

Abstract included in tex

Growth cones advance by forward translocation of the C-domain and axonal framework <i>in</i><i>vitro</i>.

<p>(<b>A</b>) Phase and (<b>B</b>) fluorescent images over 1 h of MitoTracker labelled <i>Drosophila</i> neurons grown on poly-ornithine. Kymographs of the phase images (<b>C</b>) and fluorescent images (<b>D</b>) show the position of the growth cone and mitochondria over time. (<b>E</b>) Green arrows overlaid on the kymograph illustrate the movement of docked mitochondria and the blue arrows show the tracks of fast transported mitochondria. The corresponding images from a neuron grown on DECM are shown in panels (<b>F</b>-<b>J</b>). Time arrow = 30 min and scale bar is 10 µm for both the time-lapse images and kymographs. (<b>K</b>) Quantitative analysis of the velocity of docked mitochondria plotted against distance from the growth cone. Errors bars are 95% confidence intervals. The numbers at the base of the bars denote the number of mitochondria analyzed in each bin. The growth cone is defined as the first 5 µm of axon. </p

miRNA screen and genetic validation of the requirement of miR-14 for TE repression.

<p>(A) Detection of the piRNA sensor expression following titration of two positive miRNAs by <i>tj-</i>driven expression of the corresponding miRNA-sponges, miR-14SP (middle) and miR-34SP (right). After 1h of β-Gal staining no staining was observed in the sibling ovaries without any miRNA-sponge (Ø), as illustrated by the control for the miR-14SP experiment (left). At that time, only de-repression of the <i>gypsy-</i>lacZ reporter gene, but not yet of the <i>ZAM-</i>lacZ reporter gene, could be detected. (B) Fold changes in the steady-state RNA levels of the lacZ reporters (see primer sequence in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005194#pgen.1005194.s009" target="_blank">S3 Table</a>) upon miR-14SP- and miR-34SP-induced miRNA titration. Quantification was done relative to <i>RpL32</i> and normalized to sibling ovaries with no miRNA sponge (error bars represent ± SD; n = three biological replicates). (C) Fold changes in the steady-state RNA levels of three follicle cell-specific TEs (<i>ZAM</i>, <i>Tabor</i> and <i>Stalker2</i>) upon miR-14SP and miR-34SP <i>tj</i>-driven expression. Quantification was done relative to RpL32 and normalized to sibling ovaries with no miRNA-sponge (error bars represent the SD of three biological replicates). The absence of <i>Tabor</i> de-repression might indicate that the <i>Tabor</i> family lacks active elements in the tested genotypes. (D) Fold changes in the steady-state level of the three major <i>ZAM</i>, <i>Tabor</i> and <i>traffic jam</i> (<i>tj</i>) piRNAs (see sequences in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005194#pgen.1005194.s009" target="_blank">S3 Table</a>), upon miR-14SP- and miR-34SP-induced miRNA titration. Quantification was done relative to miR-9c and normalized to sibling ovaries with no miRNA sponge (error bars represent the S.D. of three biological replicates). (E) Fold changes in miRNA and piRNA levels induced by the miR-14 null mutation (ΔmiR-14). Quantification was done relative to miR-989 and normalized to heterozygous sibling ovaries. ul: undetectable level (error bars represent the SD of three biological replicates).</p

Follicle cell-specific defects in the miRNA pathway lead to <i>ZAM-</i>lacZ reporter and somatic TE de-repression.

<p>(A) Comparison of <i>ZAM</i>-lacZ reporter expression in control ovaries in which WT-Drosha expression is driven by the <i>tj-</i>GAL4 somatic driver (<i>tj</i>-GAL4>WT-Drosha) or that contain two independent hairpins against Drosha without any driver (Ø><i>drosha-IR°</i>) and in ovaries in which <i>tj-</i>GAL4 drives the expression of the trans-dominant negative Drosha construct (<i>tj</i>-GAL4>TN-Drosha) or the two Drosha long hairpins (<i>tj</i>-GAL4><i>drosha-IR°</i>). The blue β-Gal staining is shown in black. (B) Comparison of <i>ZAM-</i>lacZ reporter expression in ovaries where <i>gawky</i> (<i>tj</i>-GAL4>sh<i>gawky</i>) or <i>AGO1</i> (<i>tj-GAL4</i>>sh<i>AGO1</i>) were silenced by <i>tj</i>-GAL4-induced shRNA expression, and in ovaries from the respective sibling controls (<i>tj</i>-GAL4>Ø). The blue β-Gal staining is shown in black. (C) Fold changes in the steady-state RNA levels of the <i>ZAM</i>-lacZ reporter (PCR1 primer pair, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005194#pgen.1005194.s009" target="_blank">S3 Table</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005194#pgen.1005194.g002" target="_blank">Fig 2A</a>), the somatic TEs <i>ZAM</i> and <i>Tabor</i> and the germline-specific TE <i>F-element</i>, following the expression, in follicle cells, of the trans-dominant negative Drosha construct (<i>tj</i>-GAL4>TN-Drosha), the <i>Drosha</i> long hairpins (<i>tj</i>-GAL4><i>drosha-IR</i>) or the <i>AGO1</i> small hairpin (<i>tj</i>-GAL4><i>shAGO1</i>). In <i>tj</i>-GAL4>Drosha-IR ovaries, the <i>ZAM</i>-lacZ reporter was replaced by the <i>UAS-dcr2</i> transgene (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005194#pgen.1005194.s008" target="_blank">S2 Table</a>). Quantification was done relative to <i>RpL32</i> and normalized to the respective controls (<i>tj</i>-GAL4>Ø, Ø><i>drosha-IR</i> and <i>tj</i>-GAL4><i>shAGO3</i>) (error bars represent the standard deviation (S.D.) of three biological replicates, log2 scale).</p

Plasma membrane signaling might be involved in miRNA-dependent TE repression.

<p>GO term comparisons of miR-14 and miR-34 putative targets and of all the Drosophila miRNA putative targets. The list of genes included in GO terms 0005886 and 0016020 is in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005194#pgen.1005194.s012" target="_blank">S6 Table</a>.</p

An LQG approach to self-tuning control with application to robotics

Self-tuning control has been recognised as an effective approach for mechanical manipulator control design due to its ability to cope with the presence of nonlinearities and uncertainties in robot dynamic models. The vast majority of existing self-tuning controllers are based on a linear plant description, the fact that most industrial processes are nonlinear is taken into account by regarding the plant as a sequence of pseudolinear descriptions. Therefore, as the plant operating point changes, the nonlinear plant dynamics are reflected as time varying parameters in the linear plant description. The applicability of this approach has been demonstrated by Koivo and Guo [1] where manipulator joint angular position is the controlled variable. This work is extended in Koivo et al [2] to the case in which the manipulator is controlled directly in the cartesian coordinate system. It is found that convergence of the parameter estimates may not be achieved during the finite time over which the· motion takes place. Therefore this approach is particularly suited to repetitive tasks where the last estimates from the previous run can be used as the initial estimates. Lelic and Wellstead [3] have successfully applied generalised pole placement to the control of a 5 axis electrically actuated robot-manipulator

<i>dNf1</i> systemic growth related RAS/ERK and cAMP/PKA signals appear functionally and topographically distinct.

<p>(A) The elevated larval CNS pERK level of <i>dNf1</i> mutants is reduced by neuronal expression of <i>dNf1</i>, but not by neuronal or heat-shock induced ubiquitous expression of PKA*. Western blot of pERK levels in larval CNS of the indicated genotypes. In lane 6, larvae received a daily 20 min 37°C heat shock throughout development, a protocol that suppresses the <i>dNf1</i> growth defect <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003958#pgen.1003958-The1" target="_blank">[4]</a>. (B) Structure of <i>UAS-PKA*</i> transgenes with 1 to 5 UAS elements. The lethality of these transgenes when driven with either <i>Ac5C-Gal4</i> or <i>elav-Gal4</i> is indicated by † whereas (−) indicates viable offspring. (C) Western blot of adult head lysates showing relative expression of <i>GMR-Gal4</i>-driven transgenic PKA*. Tubulin is used as a loading control. (D) Expression of PKA* or knockdown of <i>dnc</i> by shRNAi in the ring gland rescues the <i>dNf1</i> pupal size defect. In contrast, <i>UAS-dNf1</i> expression with the same ring gland drivers fails to restore systemic growth. (E–H) Expression pattern of <i>Akh-Gal4</i> driving <i>UAS-GFP</i>, co-stained with DAPI and anti-dNF1. GFP expression in the corpora cardiaca (CC) is indicated. Scale bar = 50 µm. As previously noted <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003958#pgen.1003958-Walker2" target="_blank">[74]</a>, anti-dNf1 staining is strong in the CNS, whereas staining in the ring gland is close to background.</p

miR-132 is expressed in newborn SVZ neurons at the onset of synaptic integration.

<p>(<b>A–D</b>) <i>In situ</i> hybridization images of miR-132 with TOPRO-3 (red) overlay (red, A), miR-132 (B), miR-1 (C), and miR-9 (D) in a sagittal section containing the SVZ, RMS and OB. (<b>E–G</b>) Higher magnification of miR-132, miR-1 and miR-9 images in the granule cell layer (GCL). Scale bars: 100 µm (A–D) and 30 µm (E–F). The image in (E) comes from the boxed region in (A). (<b>H</b>) Bar graphs of miR-132 qRT-PCR fold changes in the RMS_OB and GCL compared to the SVZ.</p