Normal synaptic transmission, but reduced spontaneous release in the mutants.

<p>(A,E) Five representative traces showing evoked excitatory junction potentials (EJPs) evoked by 1 Hz stimulation and low extracellular calcium, recorded from muscle 4, 6 or 7 of wild-type (yw), <i>spz</i>² and <i>DNT1</i>^<i>55</i><i>DNT2</i>^{<i>e03444</i>} mutant third instar larvae. Loss of <i>spz, DNT1</i> and <i>DNT2</i> has no effect on either the time course (A,E) or the amplitude (D,H) of the response. (B,F) Four representative traces of recordings of spontaneous neurotransmitter release (mEJPs). (C,G) The frequency and amplitude of the spontaneous mEJPs recorded from muscle 4 are both reduced in <i>spz</i>² mutants; quantal content is not affected (D). Spontaneous mEJPs are not affected in muscle 6,7 (G). Student-t tests for pair-wise comparisons, *p<0.05, **p<0.01, ***p<0.001. For detailed genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s001" target="_blank">Table S1</a> and for statistical details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s002" target="_blank">Table S2</a>.</p

Loss of DNT function causes a reduction in active zones.

<p>(A,B) Third instar larval NMJ preparations showing active zones labelled with nc82/Brp (green) over HRP+ axons (magenta). (B) There is a reduction in active zones per bouton in the mutants (higher magnification details from A). (C, E) Number of active zones per bouton: (C) counted manually and (E) using DeadEsy Synapse (as total nc82/Brp voxel occupancy per bouton), one bouton was chosen randomly from each NMJ. (D, F) Number of active zones per NMJ terminal: (D) counted manually and (F) active zone density per NMJ terminal, measured using DeadEasy Synapse (as total nc82/Brp voxel occupancy per terminal), decreases in the mutants. (G) Rescue: the reduction in active zone number per bouton and DeadEasy voxel volume per bouton at the muscle 6,7 NMJ in <i>DNT1</i>^<i>41</i><i>DNT2</i>^{<i>e03444</i>} double mutants is rescued with the over-expression of the <i>DNTs</i> in neurons. (H) Rescue: the number of active zones per NMJ terminal and of total active zone occupancy in the 6,7 NMJ in <i>DNT1</i>^<i>41</i><i>DNT2</i>^{<i>e03444</i>} double mutants is rescued with the over-expression of the <i>DNTs</i> in neurons. Numbers within bars indicate n=number of NMJs analysed. Asterisks indicate comparisons to <i>yw</i> control, all samples were analysed using One Way ANOVA and post-hoc Dunnett tests: *p<0.05, **p<0.01, ***p<0.001. Scale bars: 20 µm. For raw data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone-0075902-t001" target="_blank">Table 1</a>, for detailed genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s001" target="_blank">Table S1</a> and for statistical details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s002" target="_blank">Table S2</a>.</p

Loss and gain of DNT function induce changes in bouton morphology and number and axonal terminal length at the NMJ.

<p>(A,C, F) Third instar larval NMJ preparations, showing synaptic boutons by the colocalisation of pre-synaptic HRP (magenta, also labelling the axons) and post-synaptic Dlg (green). (A’, C’) High magnification details of (A,C) showing an increase in bouton number (A’, <i>spz</i>²), and stretches of Dlg signal along the axons with no boutons (A’ <i>spzCK</i>, C’) (B,D) Quantification of bouton number normalized to muscle surface area (MSA). (E-G) Axonal terminal length increases upon alterations in DNT function, (F) shows the muscle 6,7 NMJ; quantification in (E,G) shows axonal terminal length normalized over MSA. Numbers within bars indicate n=number of NMJs analysed. Significance: (B,D) One Way ANOVA: p=0.000 and p=0.001, and post-hoc Dunnett corrections. (E) One Way ANOVA: p=0.029. (G) One Way ANOVA: p<0.000. Asterisks indicate comparisons to control yw, Dunnett post-hoc tests, * p<0.05, **p<0.01, ***p<0.001. GAL4 drivers: <i>elavGAL4</i> (neurons) or <i>24BGAL4</i> (muscle). Scale bars: 20 µm. For raw data see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone-0075902-t001" target="_blank">Table 1</a>, for detailed genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s001" target="_blank">Table S1</a> and for statistical details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s002" target="_blank">Table S2</a>.</p

The DNTs have neuronal functions that are essential for viability and are redundant.

<p>Survival index is the ratio of homozygous to heterozygous pupae, relative to the Mendelian expectation for wild-type viability, given by S.I. = 2x No. Homozygous Tb⁺ /No.TM6B Tb^-. For wild-type, S.I.=1. <i>elavGAL4</i> drives expression in all neurons and <i>24BGAL4</i> in all muscles. (A) Rescue of <i>spz</i>² semi-lethality expressing <i>p35, activated </i><i>spzCK, DNT1CK3</i>’<i>+, DNT2CK</i> or <i>Toll</i>^<i>10b</i> in neurons. Note that expression of activated <i>spzCK</i> (arrow) and <i>Toll</i>^<i>10b</i> in neurons rescues the <i>spz</i>² lethality, expression of <i>DNT2CK</i> in muscle also rescues (arrow), but expression of <i>Toll</i>^<i>10b</i> in muscle does not (arrow). (B) Rescue of <i>DNT1</i>^<i>55</i><i>DNT2</i>^{<i>e03444</i>} double mutant semi-lethality at 18°C by <i>p35, DNT1CK3</i>’<i>+, DNT2CK, spzCK</i> and <i>dTRAF2</i> over-expression in neurons. Note that expression of only <i>DNT1CK3</i>’<i>+</i> or <i>DNT2CK</i> in neurons partially rescue the semi-lethality of the double mutant (arrow), indicating that they have distinct functions. Activated <i>spzCK</i> can also rescue the lethality of the double mutants, indicating redundant functions (arrow). <i>Elav-p35</i> is a fusion of the elav promoter upstream of p35, independently of GAL4. GAL4 drivers: <i>w;elavGAL4</i> (all neurons), <i>w;chaGAL4</i> (cholinergic neurons) or <i>w; 24BGAL4</i> (muscles). Numbers above bars indicate sample sizes n=number of pupae scored. Statistical tests: Chi-square with Bonferroni correction, *p<0.05, **p<0.01, ***p<0.001. For detailed genotypes see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s001" target="_blank">Table S1</a> and for statistical details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075902#pone.0075902.s002" target="_blank">Table S2</a>.</p

Neuron-Type Specific Functions of DNT1, DNT2 and Spz at the Drosophila Neuromuscular Junction

<div><p>Retrograde growth factors regulating synaptic plasticity at the neuromuscular junction (NMJ) in Drosophila have long been predicted but their discovery has been scarce. In vertebrates, such retrograde factors produced by the muscle include GDNF and the neurotrophins (NT: NGF, BDNF, NT3 and NT4). NT superfamily members have been identified throughout the invertebrates, but so far no functional <i>in vivo</i> analysis has been carried out at the NMJ in invertebrates. The NT family of proteins in Drosophila is formed of DNT1, DNT2 and Spätzle (Spz), with sequence, structural and functional conservation relative to mammalian NTs. Here, we investigate the functions of Drosophila NTs (DNTs) at the larval NMJ. All three DNTs are expressed in larval body wall muscles, targets for motor-neurons. Over-expression of DNTs in neurons, or the activated form of the Spz receptor, <i>Toll</i>^<i>10b</i>, in neurons only, rescued the semi-lethality of <i>spz</i>² and <i>DNT1</i>^<i>41</i><i>, DNT2</i>^{<i>e03444</i>} double mutants, indicating retrograde functions in neurons. In <i>spz</i>² mutants, <i>DNT1</i>^<i>41</i><i>, DNT2</i>^{<i>e03444</i>} double mutants, and upon over-expression of the DNTs, NMJ size and bouton number increased. Boutons were morphologically abnormal. Mutations in <i>spz</i> and <i>DNT1,DNT2</i> resulted in decreased number of active zones per bouton and decreased active zone density per terminal. Alterations in DNT function induced ghost boutons and synaptic debris. Evoked junction potentials were normal in <i>spz</i>² mutants and <i>DNT1</i>^<i>41</i><i>, DNT2</i>^{<i>e03444</i>} double mutants, but frequency and amplitude of spontaneous events were reduced in <i>spz</i>² mutants suggesting defective neurotransmission. Our data indicate that DNTs are produced in muscle and are required in neurons for synaptogenesis. Most likely alterations in DNT function and synapse formation induce NMJ plasticity leading to homeostatic adjustments that increase terminal size restoring overall synaptic transmission. Data suggest that Spz functions with neuron-type specificity at the muscle 4 NMJ, and DNT1 and DNT2 function together at the muscles 6,7 NMJ.</p> </div

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

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

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

Retrograde DNT2 binds pre-synaptic Kek-6 activating CaMKII and regulating structural synaptic plasticity.

<p>(A) Illustration of Kek-6 compared to Trk isoforms. DNT2 binds Kek-6, which functions via CaMKII and VAP33A downstream. (B) Pre-synaptic motoneuron terminal at the NMJ: DNT2 is produced at the muscle and secreted, binds pre-synaptic Kek-6, functioning via CaMKII and VAP33A downstream. DNT2 also binds Toll-6 which can interact with Toll-6. (C) The concerted functions of DNT2 and its two receptors Kek-6 and Toll-6 regulates NMJ growth and synaptic structure. Kek-6 functions via CaMKII and VAP33A downstream, the mechanism downstream of Toll-6 at the NMJ has not been investigated in this work. Red arrows: positive regulation by Kek-6; blue arrows: positive regulation by Toll-6. (D-F) Summary of the experimental evidence provided, green arrows indicate up- or down-regulation as a result or loss or gain function genotypes. Altering the levels of DNT2, Kek-6 and Toll-6 affects locomotion, NMJ growth and synaptic structure. Importantly, loss of both kek-6 and Toll-6 prevents homeostatic compensation of active zones, and whereas gain of function for kek-6 or Toll-6 is not sufficient to increase NMJ size, over-expression of DNT2 can. The data suggest that Kek-6 and Toll-6 function in concert as a receptor complex for DNT2, to regulate structural synaptic plasticity.</p

Kek-6 functions downstream of DNT2.

<p>Confocal images of NMJs from A3-4 muscle 6/7 (left), and box-plot graphs (right), showing: (A) Over-expression of <i>kek-6</i> in motoneurons rescued the phenotypes of <i>kek-6</i> mutants. Dlg: One Way ANOVA p<0.0001 and post-hoc Bonferroni *p<0.05, ***p<0.001. HRP: One Way ANOVA p<0.001 and post-hoc Bonferroni **p<0.01, **p<0.01. (B) Left: Over-expression of <i>kek-6</i> in neurons rescued the phenotype of <i>DNT2</i> mutants. Dlg: Kruskal-Wallis p = 0.001, and post-hoc Dunn test **p<0.01, ***p<0.001. Right: <i>kek-6</i> loss of function rescued the increase in boutons caused by the muscle over-expression of <i>DNT2</i>. Dlg: Welch ANOVA p<0.01 and post-hoc Games-Howell *p<0.05, **p<0.01. (C) Over-expression of <i>kek-6</i> in motoneurons rescued the phenotypes of <i>kek-6 DNT-2</i> double mutants. Dlg: Kruskal-Wallis p = 0.001 and post-hoc Dunn’s test *p<0.05, **p<0.01. HRP: Welch ANOVA p = 0.000, post-hoc Games Howell **p<0.01. n = 29–101 hemisegments. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.s006" target="_blank">S1 Table</a>. GAL4 drivers: Muscle: <i>MhcGAL4</i>; Neurons: <i>elavGAL4;</i> MN: <i>D42 or Toll-7GAL4</i>. Controls: white boxes: yw/+; grey boxes: GAL4/+; mutant genotypes as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006968#pgen.1006968.g004" target="_blank">Fig 4</a>. Rescue genotypes: (A) <i>w; UASkek6RFP/+; Df(3R)6361/kek6</i>^<i>34</i><i>D42GAL4</i>. (B) <i>w; UASkek6RFP/+; elavGAL4 Df(3L)6092/ DNT2</i>^<i>37</i>; and <i>w; UASDNT2-FL/+; Df(3R)6361/kek6</i>^<i>34</i><i>D42GAL4</i>. (C) <i>w; Toll-7GAL4/UASkek6RFP; kek6</i>^<i>34</i><i>Df(3L)6092/ Df(3R)6361 DNT2</i>^<i>37</i>.</p