VD/DD motor neurons and axons in <i>C</i>. <i>elegans</i>.

<p><b>(A)</b> Diagram of an early L2 larval <i>C</i>. <i>elegans</i> hermaphrodite highlighting the position and structure of the DD motor neurons (red) and axons (black). Anterior is to the left, and dorsal is up. The blue lines represent the ventral and dorsal muscle quadrants. In the early L2 larval stage, the VD neurons (green) extend axons anteriorly in the ventral nerve cord after which the axons turn dorsally and migrate to the dorsal nerve cord to form commissures. Only two of the 13 VD neurons are shown. While migrating towards the dorsal nerve cord, VD growth cones display an extended, protrusive morphology with highly dynamic filopodial protrusions (VD8). VD7 shows the final structure of the VD neurite. <b>(B)</b> Fluorescent micrograph of an early L2 larval wild-type commissure indicated by an arrow, and a VD growth cone indicated by an arrowhead. CB, cell body; DNC, dorsal nerve cord; and VNC, ventral nerve cord. Scale bar represents 5μm. <b>(C)</b> Diagram of an L4 hermaphrodite after all the VD axon outgrowth is complete. The 18 commissures on the right side of the animal are shown (black lines), and axon guidance defects of these commissures were scored. One commissure (VD1) extends on the right side and was not scored. Of the 18 commissures on the right side, two (DD1 and VD2) extend as a single fascicle. Others pairs occasionally extended as single fascicles as well, resulting in an average of 16 observable commissures per <i>wild-type</i> animal.</p

FMO-5 can inhibit growth cone filopodial protrusion.

<p><b>(A</b>) Rescue of <i>fmo-5(tm2438)</i> VD/DD axons by transgenes containing genomic <i>fmo-5</i> (<i>Ex[fmo-5 genomic]</i>). Data for transgenic arrays are the combined results from three independently-derived arrays with similar effects. Single asterisks (*) indicate a significant difference between wild type and the mutant (<i>p</i> < 0.001); Double asterisks (**) indicates a significant difference between the mutant and rescuing transgene (<i>p</i> < 0.001) determined by two-sided <i>t</i>-test with unequal variance. <b>(B)</b> Rescue of <i>fmo-5(tm2438)</i> VD growth cone filopodial protrusions by transgenes containing genomic <i>fmo-5</i> (<i>Ex[fmo-5 genomic]</i>). Data for transgenic arrays are the combined results from three independently-derived arrays with similar effects. Average lengths of filopodial protrusions are shown (μm). Error bars represent 2x standard error of the mean. Single asterisks (*) indicate a significant difference between wild type and the mutant (<i>p</i> < 0.001); Double asterisks (**) indicates a significant difference between the mutant and rescuing transgene (<i>p</i> < 0.001) determined by two-sided <i>t</i>-test with unequal variance <b>(C-E)</b> Fluorescence micrographs of VD growth cones in <i>wild-type</i>, <i>fmo-5(tm2438)</i>, and <i>fmo-5(tm2438); Ex[fmo-5 genomic]</i>. Arrows indicate representative filopodia. Scale bar: 5μm.</p

FMO-1, FMO-4 and EHBP-1 are required for Rac GTPase-mediated inhibition of VD growth cone protrusion.

<p><b>(A,B)</b> Quantification of VD growth cone filopodial length and growth cone area in <i>wild-type</i>, activated <i>ced-12(G12V)</i> and <i>mig-2(G16V)</i>, and double mutants. (A) Average filopodial length, in μm. (B) Growth cone area in μm². Error bars represent 2x standard error of the mean. Asterisks indicate significant difference between <i>ced-10(G12V) mig-2(G16V)</i> and their respective double mutants (*<i>p</i> < 0.05, **<i>p</i> < 0.001) determined by two-sided <i>t</i>-test with unequal variance. n.s., not significant. <b>(C-F)</b> Representative fluorescent micrographs of mutant VD growth cones. (C,D) Images of <i>ced-10(G12V)</i> and <i>fmo-4(ok294); ced-10(G12V)</i> growth cones. The arrowhead in (C) points to a growth cone with limited protrusion, and the arrow in (D) indicates a filopodial protrusion. (E,F) Images of <i>mig-2(G16V)</i> and <i>fmo-5(tm2438); mig-2(G16V)</i> growth cones. The arrowhead in (E) points to a growth cone with limited protrusion, and the arrow in (F) indicates a filopodial protrusion. Scale bar: 5μm.</p

Mutations in <i>fmo-1</i>, <i>fmo-4</i>, <i>fmo-5</i> and <i>ehbp-1</i> cause axon pathfinding defects.

<p><b>(A)</b> Percentage of VD/DD axons with pathfinding defects (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006998#sec002" target="_blank">Materials and Methods</a>) in single mutants, double mutants and triple mutant harboring the <i>juIs76[Punc-25</i>::<i>gfp]</i> transgene. Single asterisks (*) indicate the significant difference between wild-type and the mutant phenotype (<i>p</i> < 0.01); Double asterisks (**) indicate significant difference between double mutants and the predicted additive effect of single mutants (<i>p</i> < 0.01) determined by Fischer’s exact test. Error bars represent 2x standard error of proportion. <b>(B-D)</b> Representative fluorescent micrograph of L4 VD/DD axons. Anterior is to the left, and dorsal is up. The scale bar represents 5μm. DNC, dorsal nerve cord; and VNC, ventral nerve cord. (B) A <i>wild-type</i> commissure is indicated by an arrow. (C) An <i>fmo-1(ok405)</i> commissure branched and failed to reach to dorsal nerve cord (arrow). (D) <i>fmo-5(tm2438)</i> VD/DD axons branched and wandered (arrows). A gap in the dorsal nerve cord (asterisk) indicates that commissural processes failed to reach the dorsal nerve cord. determined by Fischer’s exact test. At least 1500 axons were scored per genotype. M+ indicates that the animal has wild-type maternal <i>ehbp-1(+)</i> activity.</p

FMO-1, FMO-4, FMO-5, and EHBP-1 are required for MYR::UNC-40-mediated inhibition of VD growth cone protrusion.

<p><b>(A,B)</b> Quantification of VD growth cone filopodial length and growth cone area in <i>wild-type</i>, <i>myr</i>::<i>unc-40 (lqIs128</i> and <i>lqIs129)</i> and double mutants. (A) Average filopodial length, in μm. (B) Growth cone area in μm². Error bars represent 2x standard error of the mean. Asterisks indicate significant difference between <i>myr</i>::<i>unc-40</i>, <i>wild-type</i> and the double mutants (*<i>p</i> < 0.05, ** <i>p</i> < 0.001) determined by two-sided <i>t</i>-test with unequal variance. <b>(C-E)</b> Fluorescent micrographs of mutant VD growth cones; (C) Image of a <i>myr</i>::<i>unc-40</i> growth cone in an early L2 animal. The arrowhead points to a growth cone with little or no filopodial protrusion. (D, E) Images of <i>fmo-4(ok294); myr</i>::<i>unc-40</i> and <i>fmo-5(tm2438); myr</i>::<i>unc-40</i> growth cones. Filopodial protrusions are indicated (arrows). Scale bar: 5μm. <i>fmo-1(ok405); myr</i>::<i>unc-40</i> double mutants were built and compared with <i>lqIs129[myr</i>::<i>unc-40]</i> due to the linkage of the <i>lqIs128</i> transgene.</p

Expression of <i>fmo-1</i>, <i>fmo-4 and fmo-5</i> in VD/DD neurons rescues axon pathfinding defects.

<p><b>(A)</b> The percentages of VD/DD axons failing to cross the lateral mid-line are as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006998#pgen.1006998.g005" target="_blank">Fig 5A</a>. <i>unc-5</i> double mutant genotypes are indicated, and the <i>Punc-25</i>::<i>fmo-1</i>, <i>Punc-25</i>::<i>fmo-4</i>, and <i>Punc-25</i>::<i>fmo-5</i> transgenes are bracketed. Data for transgenic strains are the combined results from three independently-derived transgenes with similar effects. Double asterisks (**) indicate a significant difference between the double mutant and the transgenic strain (<i>p</i> <0.001; Fisher’s exact test). Error bars represent 2x standard error of the proportion. <b>(B, C)</b> Micrographs of mutant and rescued animals. Dorsal is up, anterior to the left. Scale bar represents 5μm. The lateral midline of the animal is indicated by the dashed white line. The dorsal nerve cord and ventral nerve cord are indicated by dotted white lines. (B) <i>fmo-1(ok405) unc-5(e152)</i> axons often fail to cross the lateral midline (arrow). (C) <i>fmo-1(ok405) unc-5(e152</i>); Ex(<i>Punc-25</i>::<i>fmo-1</i>) axons crossed the lateral midline (arrows). <b>(D-E and D’-E’)</b> Images are micrographs of L2 animals with transgenic expression of <i>Pfmo-4</i>::<i>gfp</i> and <i>Pfmo-5</i>::<i>fmo-5</i>::<i>gfp</i>. Dorsal is up and anterior is left. Scale bar: 5μm. (D) <i>fmo-4</i>::<i>gfp</i> is broadly expressed, including in hypodermis and in cells along the ventral nerve cord that resemble motor neurons. Expression is not evident in the lateral hypodermal seam cells (asterisks). (D’) Enlarged image of <i>fmo-4</i>::<i>gfp</i> expression in ventral nerve cord cells (arrows). (E). <i>fmo-5</i>::<i>gfp</i> is expressed strongly in the gut (asterisks), as well as in cells along the ventral nerve cord. (arrow) (E’) Enlarged image of <i>fmo-5</i>::<i>gfp</i> expression in ventral nerve cord cells (arrows).</p

FMO-5 activity can partially compensate for UNC-5, UNC-73/Trio, and UNC-33/CRMP.

<p><b>(A,B)</b> Quantification of VD growth cone filopodial length and growth cone area in indicated genotypes. Error bars represent 2x standard error of the mean. Asterisks indicate significant difference between wild-type and mutants (** <i>p</i> < 0.001) and *** indicate a significant difference between each single mutant compared to the double mutant. Pound signs (<b>#</b>) indicate a significant difference between [<i>fmo-5 genomic</i>] and double mutant (#<i>p</i> < 0.001) determined by two-sided <i>t</i>-test with unequal variance. n.s., not significant. <b>(C-H)</b> Fluorescence micrographs of VD growth cones from <i>wild-type</i>, <i>fmo-5(tm2438)</i>, <i>[fmo-5 genomic]</i>, <i>unc-5; [fmo-5 genomic]</i>, <i>unc-33; [fmo-5 genomic]</i>and <i>fmo-1; [fmo-5 genomic]</i>. The arrowhead points to a growth cone with limited protrusion. Arrows indicate representative filopodia. Scale bar: 5μm.</p

<i>fmo</i> genes and <i>ehbp-1</i>.

<p><b>(A)</b> Diagram of Drosophila MICAL, the <i>C</i>. <i>elegans</i> flavin monooxygenases (FMOs), and <i>C</i>. <i>elegans</i> EHBP-1. MO, flavin monooxygenase domain; CH-calponin homology domain, CC, coiled-coiled domain; LIM, LIM domain. <b>(B)</b> The structures of the <i>fmo-1</i>, <i>fmo-2</i>, <i>fmo-3</i>, <i>fmo-4</i>, <i>fmo-5</i> and <i>ehbp-1</i> genes are shown. Filled boxes represent exons. The extent of deletions in <i>fmo-1</i>, <i>fmo-2</i>, <i>fmo-4</i>, <i>fmo-5</i> and <i>ehbp-1</i> are shown below the structure, indicated by a red line. The red arrow points to the region of the splice site mutation in <i>fmo-3</i>. Scale bar indicates 500bp.</p

Axon pathfinding defects in <i>unc-40(n324)</i> are enhanced by loss of <i>fmo-1</i>, <i>fmo-4</i> and <i>fmo-5</i>.

<p><b>(A)</b> Percentage of VD/DD axons that failed cross the lateral midline of L4 hermaphrodites. Error bars represent 2x standard error of the proportion; double asterisks (**) indicates a significant difference between <i>unc-40(n324)</i> alone and the double mutants (<i>p</i> < 0.001) determined by Fisher’s exact test. Only axon commissures visibly emanating from the ventral nerve cord were scored. <b>(B,C)</b> Representative images showing VD/DD axons (arrows) after their complete outgrowth in L4 animals. The lateral midline of the animal is indicated by the dashed white line. The dorsal nerve cord and ventral nerve cord are indicated by a dotted white line. Dorsal is up, anterior is to the left. Scale bar represents 5μm. (B) In <i>unc-40(n324)</i>, many axons extend past the lateral midline, as evidenced by axons in the dorsal nerve cord (arrowheads). (C) In <i>fmo-1(ok405); unc-40(n324</i>), an increased number of axons did not cross the midline resulting in extensive regions of dorsal nerve cord without axons (arrowheads). Arrowhead indicates large gaps in the dorsal nerve cord.</p

Expression of <i>fmo-5</i> in VD/DD neurons rescues axon pathfinding defects and growth cone filopodial protrusion.

<p><b>(A</b>) Rescue of <i>fmo-5(tm2438)</i> VD/DD axons by transgenes expressing <i>fmo-5</i> under the <i>unc-25</i> promoter (<i>Ex[Punc-25(fmo-5)]</i>). Data for transgenic arrays are the combined results from three independently-derived arrays with similar effects. Single asterisks (*) indicate a significant difference between wild type and the mutant (<i>p</i> < 0.001); Double asterisks (**) indicates a significant difference between the mutant and rescuing transgene (<i>p</i> < 0.001) determined by two-sided <i>t</i>-test with unequal variance. (<b>B</b>) Rescue of <i>fmo-5(tm2438)</i> VD growth cone filopodial length by transgenes expressing <i>fmo-5</i> under the <i>unc-25</i> promoter (<i>Ex[Punc-25(fmo-5)]</i>). Average lengths of filopodial protrusions are shown (μm). Error bars represent 2x standard error of the mean. Data for transgenic arrays are the combined results from three independently-derived arrays with similar effects. Single asterisks (*) indicate a significant difference between wild type and the mutant (<i>p</i> < 0.001); Double asterisks (**) indicates a significant difference between the mutant and rescuing transgene (<i>p</i> < 0.001) determined by two-sided <i>t</i>-test with unequal variance. n.s., not significant. <b>(C-E)</b> Fluorescence micrographs of VD growth cones in <i>fmo-5(tm2438)</i>, <i>fmo-5(tm2438); Ex[Punc-25</i>::<i>fmo-5]</i> and <i>fmo-5(tm2438); Ex[Pdpy-7</i>::<i>fmo-5]</i> which showed no rescue. Arrows indicate representative filopodia. Scale bar: 5μm.</p