Flavin monooxygenases regulate Caenorhabditis elegans axon guidance and growth cone protrusion with UNC-6/Netrin signaling and Rac GTPases

The guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5:UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5:UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL (Molecule Interacting with CasL) also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL. Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD axon guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance

RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in Caenorhabditis elegans

This work is licensed under a Creative Commons Attribution 4.0 International License.UNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions are consistent with RHO-1 and RHGF-1 acting with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.NIH R01NS040945NIH R56NS095682NIH P20GM103638NIH Office of Research Infrastructure Programs (P40 OD010440)NIH GM103418University of Kansas Center for Undergraduate Researc

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

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

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-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

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

Genetic model of inhibition of growth cone protrusion.

<p>UNC-5 homodimers and/or UNC-5:UNC-40 heterodimers act through the Rac GTP exchange factor UNC-73/Trio and the Rac GTPases, which then utilize the flavin monooxygenases and UNC-33/CRMP to inhibit protrusion. The FMOs might inhibit protrusion directly, by possibly directly oxidizing F-actin, or by promoting phosphorylation of UNC-33/CRMP.</p

Axon pathfinding defects of hypomorphic <i>unc-5</i> mutants are enhanced by loss of <i>fmo-1</i> and <i>fmo-4</i>.

<p><b>(A) and (B)</b> Quantification of VD/DD axons that failed to cross the lateral midline of L4 hermaphrodites in hypomorphic <i>unc-5(e152)</i> and <i>unc-5(op468)</i> mutants alone and in double mutant animals. Error bars represent 2x standard error of the proportion; double asterisks (**) indicates a significant difference between <i>unc-5(e152)</i> or <i>unc-5(op468)</i> alone and the double mutants (<i>p</i> < 0.001) determined by Fisher’s exact test. Only visible commissural processes emanating from the ventral nerve cord were scored. <b>(C,D)</b> Fluorescence micrographs of VD/DD axons (arrows) in L4 hermaphrodites. 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. Dorsal is up, anterior is to the left. Scale bar represents 5μm. (C) In the weak loss of function <i>unc-5(op468)</i> mutants, axons crossing the lateral midline are indicated (arrows). (D) In <i>fmo-5(tm2438); unc-5(op468</i>), some axons cross the lateral midline, but many terminate before crossing the lateral midline (arrows).</p