Genetic pathway

<p>UNC-6-ligated UNC-40 induces a polarized distribution of UNC-40 in HSN (Xu et al., 2009). Genetic analyses indicate that UNC-6 induces UNC-40 polarization through parallel pathways comprising the cytoplasmic protein UNC-53 and the receptor UNC-5. The analyses also reveal an UNC-6-independent pathway that can induce the polarized UNC-40 distribution and which can be inhibited by UNC-53 activity.</p

Genetic interactions between unc-53 and unc-6 and between unc-53 and unc-5 affect the direction of axon protrusion.

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity</p> <p> </p> <p>(A) Schematic diagram of AVM and HSN axon outgrowth. Axon outgrowth is towards ventral UNC-6 sources. (B–G) Photomicrographs of L4 stage animals showing the direction of AVM axon protrusion from the cell body. Ventral is down and anterior is to the left. Scale bar: 20 µm. In the wild-type pattern, the AVM axon ventrally protrudes toward the ventral nerve cord (B). Loss of unc-6 function causes anterior protrusion (C). Axon protrusion in the unc-53 mutants is similar to that observed in wild-type animal. (D). In unc-53(n152);unc-6(ev400) double mutants, AVM axons frequently protrudes dorsally (E), posteriorly (F), or have short extra extensions (G). (H–M) Photomicrographs of L4 stage animals showing the direction of HSN axon protrusion form the cell body. Ventral is down and anterior is to the left. Arrow indicates the PLM axon. Scale bar: 10 µm. In the wild-type pattern, 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 (H). Loss of unc-6 function causes anterior axon protrusion (I). Although most unc-53(n152) mutants have the wild-type protrusion pattern, in unc-53(n152);unc-6(ev400) mutants, HSN axons frequently protrude dorsally (J), posteriorly (K), or have extra extensions (L). Although most unc-5(e53) mutants have the wild-type protrusion pattern, in unc-53(n152);unc-5(e53) mutants, HSN axons frequently protrude anteriorly (M). PLM is not shown in J, K, L, or M because axons often terminate early in unc-53(n152) mutants.</p

Direction of axon protrusion from the HSN cell body.

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity</p

EGL-20 inhibits anterior and posterior orientation of UNC-40 asymmetric localization and the formation of axons from these sites

<p><strong></strong>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity</p> <p><br><strong></strong></p> <p>(A–D) HSN neurons expressing a functional UNC-40::GFP protein in the L3 larval stage. (E–H) HSN neurons in the L3–L4 larval transition stage expressing a marker to visualize the HSN neurons. Arrow points to HSN cell body. (I–K) HSN neurons expressing a functional MIG-10::GFP protein in the L3 larval stage. (A) In wild-type animals, the UNC-40::GFP protein is ventrally localized. (B) In unc-6mutants, the UNC-40::GFP protein is uniformly dispersed around the periphery. (C and D) In egl-20 mutants, the UNC-40::GFP protein is localized ventrally as in wild-type animals or anteriorly (C) or posteriorly (D). (E) In wild-type animals the axon has a strong bias to ventrally protrude. (F) In unc-6 mutants, the axon has a bias to form anteriorly. (G and H) In egl-20 mutants the axon has a bias to form ventrally as in wild-type animals or anteriorly (G) or posteriorly (H). (I) In wild-type animals, the MIG-10::GFP protein is ventrally localized. (J,K) In unc-6 and egl-20 mutants, the MIG-10::GFP protein is uniformly dispersed around the periphery. Images are of collapsed stacks of optical sections. Ventral is down and anterior is to the left. Scale bar: 5 µm.</p

UNC-40::GFP is localized at the sites of axon outgrowth.

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity </p> <p> </p> <p>Photomicrographs of the localization of UNC-40::GFP in the HSN neuron during the early stages of axon formation. UNC-40::GFP localization is associated with the sites of axon outgrowth. (A and B) An HSN neuron was imaged over a 1 h interval in the L2 stage. During this period a single broad leading edge forms ventrally. (A) Strong and uniform UNC-40::GFP fluorescence is observed on the left side (arrow) and at a leading ventral edge. (B) After 1 hour, strong and uniform UNC-40::GFP fluorescence is observed to the right (arrow), whereas the fluorescence on the left appears less uniform and intense. (C–E) During the L3 stage multiple neurites develop. Images show a progression of outgrowth. We note that the expression of UNC-40::GFP appears to exacerbate branching. See figure 1 in Adler et al. for a comparison to wild-type animals (Adler et al., 2006). Ventral is down and anterior is to the left. Scale bar: 5 µm.</p

Genetic interactions between unc-53 and unc-6 and between unc-53 and unc-5 affect intracellular UNC-40::GFP localization.

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity </p> <p> </p> <p>(A–H) Photomicrographs of the localization of UNC-40::GFP 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 unc-6(ev400) mutants it is evenly distributed (B). Although UNC-40::GFP is ventralized in unc-53(n152) mutants (C), in unc-53(n152); unc-6(ev400) mutants it is polarized to different sides, including dorsally (D). Although UNC-40::GFP is ventralized in unc-5(e53) mutants (E), in unc-53(n152); unc-5(e53)mutants it is more evenly distributed (F) and is similar to localization in unc-6(ev400) mutants. (G) Graph indicating the dorsal–ventral orientation of UNC-40::GFP. 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 unc-6(−) mutants and an increased dorsal bias inunc-53(n152); unc-6(ev400) mutants. (*) statistic difference (P<0.05, one-tailed Student's t-test); (n.s.), statistically, no significance. (H) 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 made, the dorsal surface was geometrically divided into three equal segments, and the total intensity of each was recorded. The effect of the mutations on UNC-40 distribution can be accessed by comparing the two graphs, for example in unc-6(ev400) mutants UNC-40 is evenly distributed as indicated by the equal dorsal-to-ventral ratio and the uniform anterior–posterior value. In contrast, UNC-40 is polarized to different sides in unc-53(n152); unc-5(e53)mutants as indicated by the increased dorsal-to-ventral ratio and the increased bias to localize at the middle of the dorsal side (red) or to the anterior (blue) or posterior (green) sides.</p

Direction of axon protrusion from the HSN cell body.

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity</p

Cell-autonomous function of mig-1

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity</p> <p> </p> <p>The gmex384(unc-86::mig-1::yfp) transgene was introduced into the mig-1(e1787) genetic background. The mig-1(e1787);gmex384(unc-86::mig-1::yfp) animals exhibit partial suppression of the ventral guidance defect. “% animals with HSN ventral guidance defects” refers to the percentage of animals with any HSN axon that did not grow out towards the ventral midline.</p

HSN axon extension in EGL-20 signaling mutants

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity</p> <p> </p> <p>(A–C) HSN axon extension at the L1 stage in wild-type,egl-20(n585), and vang-1(tm1422) animals. Whereas in wild-type animals the axon protrudes in the early L4 stage, in the mutants axon protrusions are observed at earlier stages. Ventral is down and anterior is to the left. Scale bar: 5 µm. (D) Percentage of HSN neurons with a single axon in L1 and L2 stages. A neuron was scored as having an extension if the length of the neurite was at least twice the anterior to posterior length of the cell body. Significant differences (one-tailed Student's t-test), *P<0.05; **P<0.01; ***P<0.001; error bars indicated the SEM; for each condition, n≧105 animals.</p

Genes function to inhibit anterior and posterior UNC-40 localization

<p>This data is part of a published article:</p> <p>Experimental evidence for UNC-6 (netrin) axon guidance by stochastic fluctuations of intracellular UNC-40 (DCC) outgrowth activity</p> <p> </p> <p>(A) Graph indicating the dorsal–ventral orientation of UNC-40::GFP. 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 unc-6(−) mutants. In 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 (P<0.005, t-test). (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 Materials and Methods). The measurements were taken using only the dorsal periphery in order to minimize cell shape differences. In several mutants there is a bias for anterior or posterior localization. (C) Graph indicating the dorsal–ventral orientation of MIG-10::GFP. The graph shows the average ratio of dorsal-to-ventral intensity from linescan intensity plots of the MIG-10::GFP signal around the periphery of the HSN cell. MIG-10 is ventrally localized in wild type, but the ratio is different in unc-6(−) and the mutants (P<0.005, t-test) because MIG-10 is uniformly distributed along the dorsal–ventral axis. (D) Graph indicating the anterior–posterior orientation of MIG-10::GFP. To determine orientation, line-scan intensity plots of the MIG-10::GFP signal across the dorsal periphery of the HSN cell were taken and analyzed as in Fig. 2B. There is a uniform distribution in unc-6(−) mutants and the mutants along the anterior–posterior axis. Error bars represent standard error of mean. n>15.</p