A Signaling Network for Patterning of Neuronal Connectivity in the Drosophila Brain

The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. We investigated this question in the developing adult brain of Drosophila and find that it is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern

Wnt5 Acts through Fz Receptors to Control DCN Axon Extension

<div><p>(A) Confocal section through a <i>fz3/Y; UAS::CD8GFP/+; ato-Gal4-14a/+</i> adult brain. No significant effect is observed.</p> <p>(B) Confocal section through a <i>w; UAS::CD8GFP/+; ato-Gal4-14a/fz</i> adult brain. A small, but significant reduction in the number of the axons is observed.</p> <p>(C) Confocal section through a <i>w; UAS::CD8GFP/+; ato-Gal4-14a/fz2</i> adult brain. A significant reduction in the number of the axons is observed.</p> <p>(D) Confocal section of a adult brain with a two-cell <i>Fz, Fz2,</i> MARCM DCN clone (one in each hemisphere) obtained from <i>yw,hsFLP; UAS::CD8GFP/+; ato-Gal4-14a/+; Tub-gal80, FRT2A/Fz ^h51 Fz2^C1, FRT2A</i> flies. The contralateral axons fail to extend to the medulla and instead innervate the lobula (yellow arrow). This is observed in 100% of the mutant clones examined. Red arrow shows the ipsilateral dendrites of the single DCN cell in the right hemisphere.</p> <p>(E) Confocal section through a <i>wnt5/+; ato-Gal4-14a,UAS::lacZ/fz</i> adult brain: a synergistic reduction in the number of axons compared to <i>wnt5/+</i> and <i>fz/+</i> animals is observed.</p> <p>(F) Confocal section through a <i>wnt5/+; ato-Gal4-14a,lacZ/fz2</i> showing a small, but significant difference to <i>wnt5/+</i> animals.</p> <p>(G) Quantification of the axonal extension phenotype. A total of at least ten samples were evaluated for each genotype, axons were counted from each, and the average calculated (*** <i>p</i> < 0.001; * <i>p</i> < 0.03).</p></div

The FGFR Breathless Acts Upstream of Rac1 to Control DCN Axon Number

<div><p>(A) Confocal section through a <i>hep¹/Y; UAS::CD8GFP/+; ato-Gal4-14a/btl^H852–3</i> adult brain showing normal axon extension from the lobula to the medulla and therefore suppression of the <i>hep¹</i> phenotype by reduction of <i>btl</i> levels.</p> <p>(B) Confocal section through a <i>w; UAS::CD8GFP/UAS::btlDN; ato-Gal4-14a/+</i> adult brain. A marked increase in the number of axons crossing the optic chiasm is observed.</p> <p>(C) Confocal section through a <i>w; UAS::CD8GFP/UAS::btl; ato-Gal4-14a/+</i> adult brain. A reduction in the number of axons is observed resulting in interruptions of the regular DCN axon pattern.</p> <p>(D) Confocal section through a <i>w; UAS::Rac1/UAS::btlDN; ato-Gal4-14a,UAS::lacZ/+</i> adult brain. A reduction in the number of axons is observed indicating the dominance of <i>Rac1</i> phenotype.</p> <p>(E) Confocal section through a <i>w; UAS::btl/+; ato-Gal4-14a,UAS::lacZ/UAS::RacDN</i> adult brain. A large increase in the number of axons crossing the optic chiasm is observed indicating the complete dominance of the <i>Rac1</i> phenotype.</p> <p>(F) Quantification of the axonal extension phenotypes for the genotypes shown in (A–E). A total of at least ten samples were evaluated for each genotype, axons were counted from each, and the average calculated (*** <i>p</i> < 0.001).</p></div

Rac1 Acts Upstream of JNK to Control DCN Axon Number

<div><p>(A) Confocal section through a <i>w; UAS::CD8GFP/UAS::Rac1; ato-Gal4-14a/+</i> adult brain, a reduction in the number of the axons is observed resulting in interruptions of the regular DCN axon pattern.</p> <p>(B) Confocal section through a <i>w; UAS::CD8GFP/UAS::Rac1; ato-Gal4-14a/+</i> pupal brain (P + 20%–30%). Fewer axons than normal are seen extending toward the medulla at this stage in these animals.</p> <p>(C) Confocal section through a <i>w; UAS::CD8GFP/+; ato-Gal4-14a/UAS::Rac1DN</i> adult brain. A marked increase in the number of the axons crossing the optic chiasm is observed.</p> <p>(D) Confocal section of a adult brain with a two-cell <i>Rac1</i> MARCM DCN clone (one in each hemisphere) obtained from <i>yw,hsFLP; UAS::CD8GFP/+; ato-Gal4-14a/+; Tub-gal80, FRT2A/Rac1^j11, FRT2A</i> flies. Note that the contralateral axon crosses and branches in the medulla (yellow arrow). This is observed in 87% of all <i>Rac1</i> mutant cells compared to 37% of wild-type cells. Red arrow shows the ipsilateral dendrites of the DCN cell in the right hemisphere.</p> <p>(E) Confocal section through a <i>hep¹/Y; UAS::CD8GFP/+; ato-Gal4-14a/Rac1^J10</i> adult brain showing normal axon extension from the lobula to the medulla indicating that reduction of Rac1 levels rescues the loss of axon extension in <i>hep¹</i> mutants.</p> <p>(F) Confocal section through a <i>UAS::bskDN/+; ato-Gal4-14a,lacZ/UAS::Rac1DN</i> adult brain: few axons are crossing the optic chiasm indicating a complete dominance of the <i>bsk</i> loss of function phenotype.</p> <p>(G) Quantification of the axonal extension phenotype for the genotypes shown in A, B, C, D, E, and F. A total of at least ten samples were evaluated for each genotype, axons were counted from each, and the average calculated (*** <i>p</i> < 0.001).</p></div

JNK Signaling Is Required for DCN Axon Extension

<div><p>(A) Confocal section through a <i>w; UAS::CD8GFP/+; ato-Gal4-14a/+</i>. Normal axon extension from the lobula to the medulla is observed.</p> <p>(B) Confocal section through a brain from <i>UAS::bskDN/Y; UAS::CD8GFP/+; ato-Gal4-14a/+</i> showing a reduction in the number of axons crossing the optic chiasm.</p> <p>(C) Confocal section through a <i>w; UAS::CD8GFP/+; ato-Gal4-14a/UAS::hepA</i> adult brain, a large increase in the axons crossing the optic chiasm is observed.</p> <p>(D) Confocal section through a <i>UAS::bskDN/+; UAS::CD8GFP/+; ato-Gal4-14a/+</i> at P + 20%–30% showing that the reduction of the number of axons crossing observed with <i>bskDN</i> occurs early during development.</p> <p>(E) Confocal section of a whole-mount adult brain from a <i>UAS::bskDN/hsFLP; +/CyO; UAS>CD2>GFP/ato-Gal4-14a</i> fly stained for GFP. The single DCN cell shows contralateral and ipsilateral projections. (E') High magnification of the branching in the lobula of the contralateral side showing no obvious defect in branching and in the bouton-like morphology.</p> <p>(F) Confocal section of a whole-mount adult brain showing single <i>bsk</i> MARCM mutant DCNs obtained from <i>hs-FLP; FRT40, tubGAL80/bsk², FRT40; ato-Gal4-14a,UAS::lacZ/+</i> animals with no axonal outgrowth. (F') High magnification of a single <i>bsk</i> mutant DCN.</p> <p>(G) Quantification of the axonal extension phenotype of the different genotypes shown in (A–D). A total of at least ten samples were evaluated for each genotype, axons were counted from each, and the average calculated (*** <i>p</i> < 0.001)</p></div

Dsh and Rac Modulate JNK Activation

<div><p>(A) Confocal section through a <i>w; UAS::CD8GFP/wnt2; ato-Gal4-14a/+</i> adult brain. A reduction in the number of the axons is observed.</p> <p>(B) Confocal section through a <i>w; UAS::CD8GFP/wnt4; ato-Gal4-14a/+</i> adult brain. No reduction in the number of axons is observed.</p> <p>C) Quantification of the <i>wnt2</i> and <i>wnt4</i> effects on DCN axon extension. A total of at least ten samples were evaluated for each genotype, axons were counted from each, and the average calculated (*** <i>p</i> < 0.001).</p> <p>(D) Confocal section through a <i>dsh¹/Y; ato-Gal4-14a, UAS::lacZ/UAS::RacDN</i> adult brain. An increase in the number of the axons crossing the optic chiasm is observed indicating a complete dominance of the Rac1 phenotype.</p> <p>(E) Confocal section through a <i>dsh1/Y; UAS::hep/+; ato-Gal4-14a,lacZ/+</i> adult brain. A large increase in the axons crossing the optic chiasm is observed indicating a complete dominance of the <i>hep</i> gain of function phenotype.</p> <p>(F) Confocal section through a <i>UAS::bskDN/dsh1; ato-Gal4-14a,UAS::lacZ/+</i> adult brain. Almost no axons are crossing the optic chiasm.</p> <p>(G) Confocal section through a <i>dsh1/+; UAS:: btlDN /+; ato-Gal4-14a,lacZ/+</i> adult brain. No significant difference in the number of the axons crossing the optic chiasm compared with control flies. However, there is an increase in variability as indicated by the larger error bar in (I) compared to control flies.</p> <p>(H) Heads from Canton S, <i>dsh¹/+, dsh¹/dsh¹, dsh¹/dsh¹;; RacJ11,Rac2</i>^Δ<i>FRT80,Mtl</i>^Δ adults animals were lysed, and whole-head lysates were subjected to SDS-PAGE. Phosphorylated and total JNK levels were examined by immunoblot analysis with anti-P-JNK (upper panel) and total JNK antibodies (bottom panel), respectively. (H') JNK activity is calculated as the relative amount of P-JNK to total JNK. Loss of Dsh function leads to a 25% decrease in JNK activity, which is rescued to 150% of wild-type levels by reduction of Rac levels showing mutually antagonistic effects of Dsh and Rac on JNK activity.</p> <p>(I) Quantification of the axonal extension phenotype. A total of at least ten samples were evaluated for each genotype, axons were counted from each, and the average calculated (*** <i>p</i> < 0.001).</p></div

Development of the DCN Axon Connectivity Pattern

<div><p>(A) Dorsal view of a L3 brain from a <i>w; UAS::CD8GFP/+; ato-Gal4-14a/+</i> animal. GFP expression is detected in two clusters of ~40 neurons (arrow) with a commissure bridging the two hemispheres. A bundle of axons extends from each commissure into the developing optic lobes.</p> <p>(B) Confocal section of a whole-mount adult brain from a <i>w; UAS::CD8GFP/+; ato-Gal4-14a/+</i> fly stained for GFP. Expression is detected in the two clusters (arrows). The commissure is visible, as is the elaborate innervation of the optic lobes by the DCNs.</p> <p>(C) Confocal section of a right hemisphere of a L3 brain, showing that DCNs extend a bundle of axons ventrally through the brain.</p> <p>(D) Confocal section of right hemisphere of a pupal brain (P + 0%), showing the extension of DCN axons toward the developing optic lobes.</p> <p>(E) Confocal section of a right hemisphere of a pupal brain (P + 20%) revealing DCN commissural axons shortly before they extend across the optic chiasm into the medulla.</p> <p>(F) Confocal section of a right hemisphere of a pupal brain (P + 30%). Approximately 30 axons are detected traversing the optic chiasm.</p> <p>(G) A magnification of the ventral half of a similar brain to (F). Note that there are two types of axons: thick, regularly spaced (red arrows) and thin (white arrows). Many of the thinner axons are seen retracting back toward the lobula.</p> <p>(H) Confocal section of a right hemisphere of a pupal brain (P + 45%), the thin axons crossing at P + 30% have retracted leaving 12–14 axons.</p> <p>(I) Confocal section of a right hemisphere of an adult brain; the final structure is now established with 12 axons extending toward the distal medulla.</p> <p>(J) Crossing axons between the lobula and the medulla from a four-cell DCN clone. No evidence for axonal blebbing or fragmentation is seen in the area vacated by the retracting axons (yellow rectangle).</p></div

Schematic Model Representing the Interaction and Integration of the Signals Controlling the DCN Axon Extension

<p>Our data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types.</p