Wnt5 and Drl are localized to the NMJ. (A) Confocal fluorescent images showing NMJs on muscles 6 and 7 in third instar larvae. Animals were labeled with antibodies against HRP (magenta), which recognizes presynaptic membranes and Wnt5 (green). (B) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and Drl (green). Scale bar: 20 μm. (C) High magnification view of an area from A. (D) High magnification view of an area from B. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

wnt5 mutant animals exhibit a reduction in the number of boutons and active zones. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). Scale bar: 20 μm. (B) Magnification of a portion of the NMJ shown in A. (C) Quantification of active zones density (nc82 puncta/μm², left) and bouton number (per NMJ, right). (D) Representative two-electrode voltage clamp recordings from muscle 6 of control and wnt5⁴⁰⁰ mutant third instar larvae, showing evoked endplate junctional currents (EJCs). (Right) Quantification of EJC amplitudes. (E) Representative voltage clamp recordings from control and wnt5⁴⁰⁰ third instar larvae, showing spontaneous excitatory junctional currents (mEJCs) in the muscle 6 NMJ. Quantification of mEJC frequency (middle) and amplitude (right). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

wnt5 expression in neurons rescues the mutant phenotype. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). For rescue experiments, a UAS-wnt5 transgene was expressed in the wnt5 mutant background in neurons using the elav-Gal4 driver (neuron exp.) or in muscle using the 24B-Gal4 driver (muscle exp.). Scale bar: 20 μm. (B) Quantification of the number of boutons per NMJ. (C) Quantification of active zone density (nc82 puncta/μm²). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Overexpression of wnt5 in neurons produces an overgrowth of the NMJ. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). For overexpression experiments, a UAS-wnt5 transgene was expressed ubiquitously (using Actin-Gal4, act>wnt5), or in neurons (elav>wnt5) or in muscle (24B>wnt5). Scale bar: 20 μm. (B) Quantification of the number of boutons per NMJ.

drl mutant animals exhibit a reduction in the number of boutons but not active zones. (A) Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta) and the presynaptic protein Brp (nc82, green). Scale bar: 20 μm. (B) Quantification of active zone density (nc82 puncta/μm², left) and bouton numbers (per NMJ, right). (C) Representative two-electrode voltage clamp recordings from muscle 6 of control and drl² third instar larvae, showing evoked endplate junctional currents (EJCs). (D) Quantification of EJC amplitudes. (E) Representative voltage clamp recordings from control and drl² third instar larvae, showing spontaneous excitatory junctional currents (mEJCs) in the muscle 6 NMJ. (F) Quantification of mEJC frequency (left) and amplitude (right). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Expression of drl in muscle rescues the drl mutant phenotype. (A) Confocal images showing NMJs labeled with antibodies against HRP. For rescue experiments, a UAS-drl transgene was expressed in the drl mutant background in neurons using the elav-Gal4 driver or in muscle using the 24B-Gal4 driver. Scale bar: 20 μm. (B) For overexpression experiments, a UAS-drl transgene was expressed ubiquitously (act>drl), or in neurons (elav>drl) or muscle (24B>drl). Quantification of the number of boutons per NMJ for overexpression experiments. (C) Quantification of the number of boutons and branches for the rescue experiments.

Epistatic analyses of the relationship between wnt5 and drl. (A) Confocal images showing NMJs labeled with antibodies against HRP. Scale bar: 20 μm. (B) Quantification of the number of boutons per NMJ. (C) Confocal images showing NMJs labeled with antibodies against HRP. For the epistatic analysis, the UAS-wnt5 transgene was overexpressed in neurons in the drl mutant background (drl²; elav>wnt5 and drl²/Cyo; elav>wnt5). Scale bar: 20 μm. (D). Quantification of the number of boutons per NMJ.

Normal NMJ growth requires the WIF and intracellular domains of Drl. (A). Confocal fluorescent images showing NMJs labeled with antibodies against HRP (magenta). Scale bar: 20 μm. (B) Quantification of the number of boutons per NMJ. (C) Model of Wnt5-Drl signaling at the NMJ. (i) Wnt5 signals through Drl to regulate NMJ size. (ii, iii) In the absence of either Wnt5 (ii) or Drl (iii), the NMJ is reduced in size. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]