Vascular anatomy in <i>dcr</i> mice.

<p>A–C) The vasculature of control (A) and two affected <i>dcr</i> (B, C) mice at 100 days of age was reconstructed using micro-CT imaging. A and B are projections viewed ventrally, C viewed dorsally. The reconstructions presented are projections, but data can be viewed in three dimensions and rotated to examine anatomy of the vasculature. No marked changes in vascular anatomy were seen in the mutant animals. D, E) Smaller penetrating vessels and capillaries of the brain were examined in coronal sections using a GFP transgene to label endothelial cells. D) A projection of a 50 µm vertical confocal reconstruction, the pial surface is in the upper left. E) Confocal analysis of <i>dcr</i> mice did not reveal a marked change in the small vessels of the brain. The pial surface in E is just out of the field at the top left, the autofluorescence in the lower left is the necrotic tissue in the cortex.</p

Growth and Mortality curves of <i>dcr</i> homozygous mice.

<p>A) The body weights of 20 control C57BL/6J male mice and 6 C57BL/6J <i>dcr</i>/<i>dcr</i> male mice are shown (mean ± SD). The body weights of the affected males begin to significantly diverge from controls at about 12 weeks of age. Gradual wasting is seen as the mice age. B) The survival of 11 C57BL/6J <i>dcr</i>/<i>dcr</i> mice is shown. Mice begin dying at 3 months of age. By 6 months, all eleven affected mice were dead. Control C57BL/6J mice from the same colony show no mortality in the same period. Note the number of mice in the body weight graph decreases with time due to mortality; however, at 12 weeks of age when the mutant curve diverges, all mice were alive.</p

Pathology in the brain of <i>dcr</i> mice.

<p>A) A coronal section of a <i>dcr</i> mouse indicates bilaterally symmetric loss of neurons in the lateral, inferior cerebral cortex (arrowheads). B) The tissue loss is consistent with a regional ischemia resulting in complete vacuolization of the tissue in the affected region with normal surrounding tissue. C) Other regions of the brain such as the dorsal medial cortex eventually become affected. D) Dilated blood vessels are often present in the brains of affected mice. E) Activated astrocytes in the striatum stained for GFAP indicate stress reactions in regions of the brain that do not show obvious pathology (Arrowheads). Note that E is from additional sectioning of the same sample in a region comparable to D.</p

The <i>dcr</i> interval.

<p>The <i>dcr</i> locus maps between marker <i>D9Dcr56</i> and <i>D9Dcr76</i>. The spacing megabase positions of these markers and two other markers within the interval are shown. Examples of genotypes defining the proximal (<i>D9Dcr56</i>) and distal (<i>D9Dcr76</i>) ends of the region are shown. Mice homozygous for C57BL/6 markers in the region are affected (<i>dcr/dcr</i> homozygotes) whereas mice heterozygous or homozygous for BALB/c markers through the region are unaffected. Both ends of the interval are confirmed by at least three independent recombination events. The position of the protein coding genes within the interval is shown below. Gene symbols are at the proximal end of the features. The noncoding miRNA (<i>AC117688</i>.1) and the processed transcript (<i>AC1130161</i>) are also shown.</p

The pathology of the <i>dcr</i> locus is progressive.

<p>A time course was performed examining five mice at each of five time points from 10 to 18 weeks (only four study mice survived to 18 weeks). A) At ten weeks, none of the mice showed obvious pathology in the inferior lateral cortex. B) By twelve weeks, four out of five mice showed detectable pathology in the inferior lateral cortex. Eosin pale areas and dying neurons (between the arrowheads, see also F) in the cortex were evident and were present in both hemispheres. C) By fourteen weeks of age, all mice showed marked pathology in the cortex with vacuolization of the tissue. D) At sixteen weeks of age, the affected regions were larger and included not only the cortex, but also the inferior hippocampus (see also G). E) By eighteen weeks of age, the surviving mice had extreme tissue loss and vacuolization. F) At early stages (twelve weeks) dying neurons identified by their eosin-positive cell bodies and condensed nuclei (black arrowhead) were evident abutting regions of apparently healthy neurons (white arrowhead) indicating a recent insult and consistent with the regional damage of an ischemic event. The image is a higher magnification view of the regions boxed in B, rotated 90 degrees clockwise. G) The hippocampus of sixteen week <i>dcr</i> animals also shows dying neurons in the CA regions of the hippocampus (CA3 shown) although the dentate gyrus (at right) remains relatively unaffected. H) The dorsal medial cortex is unaffected at sixteen weeks of age in all five mice examined. I) By eighteen weeks of age, the dorsal cortex is also involved, and eosin-pale, vacuolated areas are visible. Note H and I are more rostral than other sections, the affected dorsal medial cortical regions were consistently seen rostrally to the inferior lateral cortical regions (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015327#pone-0015327-g002" target="_blank">figure 2</a>).</p

Keks are Trk-like receptors expressed in the CNS.

<p>(A) Modular composition of TrkB, TrkB-T1, Dror, Otk and <i>Drosophila</i> LIGs. (B) Amongst the LIGs, Keks are closer to the Trks than any other mammalian or <i>Drosophila</i> LIGs, adapted from the phylogeny of Mandai et al.[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.ref022" target="_blank">22</a>]. (C,D) mRNA distribution in embryos: <i>CG15744</i>, <i>lambik</i> and <i>CG16974</i> are not expressed in the VNC (arrows) above background, but <i>lambik</i> is in PNS and <i>CG16974</i> in muscle precursors (arrowheads); <i>kek-1</i>, <i>kek-2</i> and <i>kek-6</i> transcripts are found in the VNC, and <i>kek5GAL4>tdTomato</i> drives expression in VNC and PNS (right) neurons. (E) Over-expression of <i>keks</i>– most prominently <i>kek2</i> and <i>6</i> -in all neurons with <i>elavGAL4</i> rescued the cold semi-lethality of <i>DNT1</i>^<i>41</i> <i>DNT2</i>^{<i>e03444</i>} double mutants, n = 52–313 pupae. Chi-square and Bonferroni multiple comparisons correction. *p<0.05, ***p<0.001. For statistical details see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>.</p

Kek-6 is expressed pre-synaptically in motoneurons and binds post-synaptic DNT2.

<p>(A) In Kek-6^GFP larval VNCs, GFP colocalises with the neuronal marker HB9 (arrows show examples). (B) Kek-6^GFP was found in third instar larval muscle 6/7 NMJ and synaptic boutons (dotted rectangle: higher magnification, right). (C) Kek-6^GFP was found in the motoneuron axonal terminal (arrows), and in pre-synaptic bouton lumen (dotted rectangle: higher magnification, right), not colocalising with the post-synaptic marker anti-Dlg (arrows).(D) Kek-6>FlyBow was localized to CNS axons and dendrites (arrows), and cell bodies of the RP3,4,5 motoneuron clusters (ventral and transverse views, arrows). (E) Illustration. (F) Kek-6>FlyBow was also distributed along the motoneuron axons, NMJ terminal (arrow) and synaptic boutons (arrows). (G-K) Over-expression of GFP tagged full-length DNT2 in muscle <i>(MhcGAL4>UAS-DNT2-FL-GFP)</i> revealed: (G) DNT2-GFP distribution within the pre-synaptic bouton lumen (arrows), boutons labeled post-synaptically with anti-Dlg; (H-K) DNT2-GFP along the motoraxon (labeled with anti-FasII) and within the pre-synaptic bouton lumen (arrows).</p

VAP33A functions downstream of Kek-6.

<p>(A) Confocal images of NMJs from segments A3-4, muscle 6/7. (B-E) Box-plot graphs. (B) <i>VAP33A</i>^<i>G0231</i> mutants have reduced bouton number, Mann-Whitney U test ***p<0.001. (C,D) Pre-synaptic over-expression of <i>VAP33A</i> rescues bouton number in (C) <i>kek-6</i>^{<i>–/–</i>}mutants and (D) <i>DNT2</i>^{<i>–/–</i>}single mutants, Kruskal-Wallis p<0.0001 and *p<0.05, ***p<0.001 post-hoc Dunn for both. (E) <i>kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^{<i>–/–</i>}double mutants rescue the bouton number phenotype caused by <i>VAP33A</i> gain of function, Kruskal-Wallis p<0.0001 and **p<0.01, ***p<0.001 post-hoc Dunn. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. N = 23–48 hemisegments. MN = motoneuron, <i>D42GAL4</i> (D) or <i>Toll-7GAL4</i> (E); Neurons = <i>elavGAL4</i>. Rescue genotypes: (C) <i>UASVAP33A/+; D42GAL4 kek6</i>^<i>34</i><i>/Df(3R)6361</i>. (D) <i>UASVAP33A/+; elavGAL4 Df(3L)6092/DNT2</i>^<i>37</i>.. (E) <i>UASVAP33A/Toll-7GAL4; kek6</i>^<i>34</i><i>Df(3L)6092/ Df(3R)6361 DNT2</i>^<i>37</i>.</p

<i>kek6</i> and <i>DNT2</i> mutants have smaller NMJs and impaired locomotion.

<p>(A) Plotted trajectories of filmed larvae, and (B) histograms of percentage frames at each speed analysed with FlyTracker. Kruskal-Wallis p<0.0001 and ***p<0.001 post-hoc Dunn test, n≥22344 frames. (C-E) Speed distribution classified into arbitrary categories. (C) Mutants spend more time at the lowest speeds than controls, generally do not crawl at the higher speeds (pale grey, left), but like controls can reach the highest speeds for a small fraction of time. (D) Wild-type larvae are hardly at speed = 0, contrary to the mutants. (E) All genotypes can achieve the highest speeds, but none spend much time crawling at these speeds. (F) NMJs (left, with higher magnification details of areas indicated by asterisks) and box-plot graphs (right) showing: <i>kek-6</i>^{<i>–/–</i>}and <i>DNT2</i>^{<i>–/–</i>}single mutants and <i>kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^<i>-/-</i>double mutants have fewer Dlg+ boutons, smaller HRP+ axonal terminals (normalized to muscle area, MSA), and less complex NMJs with reduced axonal branching. Dlg: Kruskal-Wallis p<0.0001, and *p<0.05, **p<0.01, ***p<0.001 post-hoc Dunn; HRP: One Way ANOVA p<0.0001, and **p<0.01, ***p<0.001 post-hoc Dunnett. <i>kek-6</i> and <i>DNT2</i> single mutants, but not the double mutants, have increased active zone density (Brp+/HRP+axonal length). Brp: Kruskal-Wallis p = 0.0012, and **p<0.01, ***p<0.001 Dunn’s post-hoc. <i>Kek-6</i> mutants have reduced Synapsin, Mann-Whitney U test ***p<0.001. For statistical details, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. N = 30–113 hemisegments. Mutant genotypes throughout figures: Control: <i>yw/+;</i> Mutants: <i>kek-6</i>^{<i>–/–</i>}: <i>kek6</i>^<i>34</i><i>/Df(3R)6361; DNT2</i>^{<i>–/–</i>}: <i>DNT2</i>^<i>37</i><i>/Df(3L)6092; kek-6</i>^{<i>–/–</i>}<i>DNT2</i>^{<i>–/–</i>}: <i>kek6</i>^<i>34</i><i>Df(3L)6092/</i> Df(3R)6361 <i>DNT2</i>^<i>37</i>.</p

Kek-6 and Toll-6 interact for NMJ structural homeostasis.

<p>(A) Toll-6GAL4>mCD8-GFP is distributed in FasII+ motoneuron axons (arrows) at the muscle 6/7 NMJ terminal. (B) Muscle 6/7 NMJs (left) and box-plot graphs (right) showing: <i>Toll-6</i>^{<i>MIO2127</i>}<i>/Df(3L)BSC578</i> mutants had fewer 1b boutons. <i>Toll-6</i>^{<i>–/–</i>}and <i>Toll-6</i>^{<i>MIO2127</i>}<i>Df(3R)6361/kek6</i>^<i>35</i> <i>Df(3L)BSC578</i> double mutants had smaller NMJs (HRP, Kruskal-Wallis p = 0.0001) with reduced branching, and reduced active zones (Brp, Kruskal-Wallis p = 0.0055), post-hoc Dunn for both *p<0.05, ***p<0.001. Pre-synaptic over-expression of <i>kek-6</i> in motoneurons in Toll-6^-/-mutants (<i>w; UASkek-6/+; Toll-6</i>^{<i>MIO2127</i>}<i>GAL4/ Df(3L)BSC578</i>) did not rescue NMJ size, but upregulated Brp+. Over-expression of activated <i>Toll-6</i>^<i>CY</i> did not affect NMJ size (HRP) but increased active zones. N = 34–46 hemisegments. (C) Co-immunoprecitation from co-transfected S2 cells: Precipitating Toll-6 and Toll-7 with anti-Flag brought down Kek-6 detected with anti-HA. IP: immuno-precipitation; WB: western blot; asterisk: co-IP. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. <i>MN = D42GAL4</i>. </p