Axon Protrusion from the AVM Cell Body.

<p>Numbers represent percentage value±SEM. Schematics depicts the direction of AVM axon protrusion.</p

The Muscle/epidermis Extracellular Matrix Inhibits Anterior and Posterior UNC-40 Localization in Response to UNC-6.

<p>(A) Graph indicating the dorsal-ventral orientation of UNC-40::GFP in HSN. The graph shows the average ratio of dorsal-to-ventral intensity from linescan intensity plots of the UNC-40::GFP signal around the periphery of the HSN cell. UNC-40::GFP is ventrally localized in wild-type, but the ratio is different in <i>unc-6(−)</i> and the mutants. (*) statistic difference (P<0.05, one-tailed Student’s <i>t-</i>test). Error bars represent standard error of mean. (B) Graph indicating the anterior-posterior orientation of UNC-40::GFP. To determine orientation, line-scan intensity plots of the UNC-40::GFP signal across the dorsal periphery of the HSN cell were taken, the dorsal surface was geometrically divided into three equal segments, and the total intensity of each was recorded. The percent intensity was calculated for each segment and ANOVA was used to determine if there is a significant difference between the three segments (see Experimental Procedures). The measurements were taken using only the dorsal periphery in order to minimize cell shape differences. In the mutants there is a bias for anterior or posterior localization, whereas there is a uniform distribution in <i>unc-6(−)</i> mutants and in double mutants with <i>unc-6(−)</i>.</p

Mutations delay the development of predominantly ventral neurites.

<p>(A) The percentage of HSN neurons with predominantly ventral neurites in mid-L3. Whereas in wild-type animals there are predominately ventral neurites by this stage, in the single mutants there is a delay. In all strains an axon will form in the L4 stage. Double mutants suggest that the development of predominate ventral neurites at the L3 stage in <i>ina-1</i> and <i>unc-52</i> mutants depends on UNC-6 signaling. Error bars indicated the SEM; n values are indicated above each column. Significant differences (two-tailed <i>t</i>-test), *P<0.001.</p

The directional bias during the initial protrusion of the HSN axon is altered by the mutations.

<p>Simulated random walks of 250 steps were plotted from an origin (0, 0). The walks were generated using the probabilities of outgrowth in the dorsal, ventral, anterior, and posterior direction, as listed for each mutant in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097258#pone-0097258-t001" target="_blank">Table 1</a>. See text for details. (A) Plots generated to compare the effect that UNC-52 (perlecan) has on the directional bias. In the <i>unc-52(e998)</i> mutant there is a bias for ventral outgrowth. This bias is eliminated in <i>unc-52(e998); unc-6(ev400)</i> and <i>unc-40(e1430);unc-52(e998)</i> mutants. The difference suggests that UNC-52 alters the direction bias of UNC-6 axon guidance by altering the probability of UNC-40-mediated axon outgrowth in each direction. This is consistent with the evidence that <i>unc-52(e998)</i> alters the probability of UNC-40-mediated axon outgrowth activity in each direction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097258#pone-0097258-g003" target="_blank">Figure 3</a>). The direction of the bias in the double mutants is different than that in <i>unc-6(ev400)</i> and <i>unc-40(e1430)</i> mutants. There is a strong bias for axon outgrowth in the anterior direction in <i>unc-6(ev400)</i> and <i>unc-40(e1430)</i> mutants. This outgrowth is UNC-40-independent, suggesting that the <i>unc-52(e998)</i> mutation also effects UNC-40-independent outgrowth activity in the double mutants. (B) Plots generated to compare the effect that INA-1 (integrin) has on the directional bias. In the <i>ina-1(gm39)</i> mutant there is a bias for ventral outgrowth. This ventral bias is eliminated in <i>ina-1(gm39); unc-6(ev400)</i> and <i>unc-40(e1430); ina-1(gm39)</i> mutants. The difference suggests that INA-1 alters the direction bias of UNC-6 axon guidance by altering the probability of UNC-40-mediated axon outgrowth in each direction. This is consistent with the evidence that <i>ina-1(gm39)</i> alters the probability of UNC-40-mediated axon outgrowth activity in each direction (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097258#pone-0097258-g005" target="_blank">Figure 5</a>). The direction of the bias in the double mutants is different than that in <i>unc-6(ev400)</i> and <i>unc-40(e1430)</i> mutants. There is a strong bias for axon outgrowth in the anterior direction in <i>unc-6(ev400)</i> and <i>unc-40(e1430)</i> mutants. Because the anterior outgrowth is UNC-40-independent, the result suggests that the <i>ina-1(gm39)</i> mutation must also effect UNC-40-independent outgrowth activity in the double mutants.</p

Mutations cause the development of precocious axons.

<p>(A) Percentage of HSN neurons with a distinct single axon in the L1 stage. A neuron was scored as having a distinct axon extension if the length of the protrusion was at least twice the anterior to posterior length of the cell body. (B) Percentage of HSN neurons with a distinct single axon in the L2 stage. Whereas in wild-type animals a distinct axon does not form until the early L4 stage, in the mutants axons are observed at earlier stages. The precocious effect of the <i>unc-52(e444)</i> mutation requires UNC-40 function, but not UNC-6. Error bars indicated the SEM; n values are indicated above each column. Significant differences (two-tailed <i>t</i>-test), *P<0.001; NS P = 0.6.</p

HSN Axon Outgrowth is directed into the Muscle/Epidermal Extracellular Matrix.

<p>(A) Photomicrograph of a wild-type L3 stage animal showing ventral HSN axon protrusion from the cell body. Arrow indicates the PLM axon. Scale bar: 5 µm. (B) Schematic diagram of the right ventral body wall showing the relative position of HSN axon outgrowth. The HSN neurites invade the extracellular matrix that lies between the longitudinal muscle cells and the epidermis. The longitudinal axon of the mechanosensory PLM neuron is enveloped by the epidermis and has specialized attachments to the epidermis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097258#pone.0097258-Emtage1" target="_blank">[32]</a>. The reported locations where the molecules considered in this study function are indicated. (C) Photomicrograph of a wild-type L3 stage <i>mig-15</i> animal showing posterior HSN axon protrusion from the cell body. In mutants, the axon often protrudes anteriorly or posteriorly along the dorsal side of the muscle/epidermis interface.</p

Mutations Affect Intracellular UNC-40::GFP Localization.

<p>(A–I) Photomicrographs showing examples of UNC-40::GFP localization in the HSN neuron of L2 stage larvae. Ventral is down and anterior is to the left. Scale bar: 5 µm. UNC-40::GFP is ventrally localized in the wild-type animals (A), but in <i>unc-6(ev400)</i> mutants UNC-40::GFP is evenly distributed and the cell shape is generally more irregular (B). In the mutants, UNC-40::GFP can be asymmetrically localized in the anterior (C), posterior (D) or in the anterior and posterior (E) direction. (F) Double mutants with <i>unc-6(ev400)</i> show the <i>unc-6(ev400)</i> phenotype with evenly distributed UNC-40::GFP and the more irregular cell shape.</p

Integrin Signaling Inhibits Anterior and Posterior UNC-40 Localization.

<p>(A) Graph indicating the dorsal-ventral orientation of UNC-40::GFP in HSN. The graph shows the average ratio of dorsal-to-ventral intensity from linescan intensity plots of the UNC-40::GFP signal around the periphery of the HSN cell. Wild-type animals show a strong ventral bias, whereas there is a uniform distribution in <i>unc-6(−)</i> mutants. In <i>ina-1</i> and <i>mig-15</i> mutants there is an intermediate phenotype indicating a weak bias for ventral localization, as well as enrichment for localization at other sites compared to wild-type. (B) Graph indicating the anterior-posterior orientation of UNC-40::GFP. To determine orientation, line-scan intensity plots of the UNC-40::GFP signal across the dorsal periphery of the HSN cell were taken and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097258#pone-0097258-g003" target="_blank">Figure 3B</a>. In the <i>ina-1</i> and <i>mig-15</i> mutants there is a bias for anterior or posterior localization. (C) Graph indicating the anterior-posterior orientation of UNC-40::GFP, including cases where there is significant localization at both the anterior and posterior sides of the neuron. Orientation was determined as in B. However, if the anterior and posterior were not significantly different and was greater than the middle segment it was scored as anterior-posterior (see Material and Methods). In the mutants there is a bias for localization in both the anterior and posterior direction. Error bars represent standard error of mean. n >15.</p

Mutations Affect the Direction of HSN Axon Protrusion.

<p>(A–G) Photomicrographs of L4 stage animals showing examples of the protrusion of the axon from the HSN cell body in wild-type and mutant animals. Ventral is down and anterior is to the left. Arrow indicates the PLM axon, if in the focal plane. Scale bar: 10 µm. (A) In wild-type animals, the HSN axon protrudes ventrally from the cell body. After reaching the ventral nerve chord the axon extends anteriorly and defasciculates from the cord to form synapses at the vulva. (B–E) In <i>mig-15</i> mutants, the HSN axon can protrude posterior (B), anterior and posterior (C), ventral (D) or anterior (E). Most axons in <i>mig-15</i> mutants that anteriorly or posteriorly protrude from the cell body eventually turn ventral. Only a few fail to reach the ventral nerve core (8% ±2, n = 100). (D–E) As axons cross the muscle/epidermis interface (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097258#pone-0097258-g001" target="_blank">Figure 1B</a>) they sometimes split into two parallel processes (26% ±1, n = 100). (F–I) As in <i>mig-15</i> mutants, the axon protrudes anteriorly, posteriorly, or ventrally in <i>unc-52</i>, <i>ina-1</i>, <i>unc-97</i>, <i>unc-112,</i> and <i>vab-19</i> mutants. In double mutants with either <i>unc-40(e1430)</i> (G) or <i>unc-6(ev400)</i> (I) the axon will sometimes protrude in the dorsal direction.</p

Axon Guidance as a Random Walk, 2014 CSHL Meeting Poster

<p>Poster presented at the 2014 CSHL meeting "Axon Guidance, Synapse Formation & Regeneration"</p> <p> </p