PMC

Highly elevated HSPG co-receptor levels at dfmr1 null NMJs. (A) Representative NMJ images co-labeled with neuronal marker anti-horseradish peroxidase (HRP, red) and anti-Dlp (Dlp, green) in control (w¹¹¹⁸) and dfmr1 null (dfmr1^50M) wandering third instar muscle 6/7. Right panels show synaptic boutons in higher magnification. Scale bars: 25 μm and 5 μm, respectively. (B) Quantification of Dlp intensity normalized to genetic controls (w¹¹¹⁸) in two dfmr1 null mutants (dfmr1^50M, dfmr1²). Sample sizes are ≥12 animals and ≥24 NMJs for each genotype. (C) Representative NMJ images co-labeled with neuronal marker (HRP, red) and anti-Sdc (Sdc, green) in w¹¹¹⁸ and dfmr1^50M flies. Scale bars: 25 μm and 5 μm (higher magnification). (D) Quantification of Sdc intensity levels. Sample sizes are ≥17 animals and ≥34 NMJs for each genotype. Significance is shown as **P<0.01 and ***P<0.001.

Restoration of dfmr1 null synaptic architecture defects by HSPG reduction. (A) Representative muscle 4 NMJ images co-labeled with presynaptic marker anti-HRP (red) and postsynaptic marker anti-Discs-large (DLG, green) in control (w¹¹¹⁸), dfmr1-null (dfmr1^50M), postsynaptic Dlp overexpression (24B × UAS-Dlp), genetic reduction of Dlp in dfmr1 null background (dlp^A18, dfmr1^50M/dfmr1^50M), and genetic reduction of both Sdc and Dlp in dfmr1 null background (sdc²³/+; dlp^A187, dfmr1^50M/ dfmr1^50M). Black/white images are shown to better highlight differences. Scale bar: 10 μm. (B,C) Quantification of total NMJ branches (B) and type 1 bouton number (C) in dfmr1 null, Dlp overexpression, and HSPG genetic reductions conditions, normalized to genetic controls. Sample size is ≥4 animals and ≥8 NMJs for each indicated genotype. Significance is shown as *P<0.05, **P<0.01 and ***P<0.001.

Increased Wg levels and spatial distribution at dfmr1 null NMJs. (A) Representative NMJ images co-labeled with neuronal marker (HRP, red) and anti-Wingless (Wg, green) in control (w¹¹¹⁸) and dfmr1 null (dfmr1^50M) wandering third instar muscle 4. Right panels show synaptic boutons at higher magnifications. Scale bars: 15 μm and 3 μm, respectively. Arrows indicate boutons that have been magnified. (B) Quantification of Wg intensity in dfmr1 nulls normalized to w¹¹¹⁸ controls. (C) Analysis of Wg spatial distribution measured as a fraction of the total HRP-labeled NMJ area. (D) Fraction of total HRP-labeled NMJ boutons expressing Wg. Samples sizes are ≥26 animals and ≥52 NMJs for each genotype. Significance is shown as *P<0.05 and ***P<0.001.

Loss of Fz2 C-terminus nuclear translocation signaling at dfmr1 null NMJs. (A) Representative muscle 4 images co-labeled with nuclear marker propidium iodide (PI, red) and anti-Frizzled C-terminus (Fz2-C, green) in control (w¹¹¹⁸) and dfmr1 null (dfmr1^50M) wandering third instars. Arrows indicate Fz2-C marked nuclei in controls. Scale bar: 20 μm. (B) Higher-magnification images of individual nuclei co-labeled with nuclear marker (PI, red) and anti-Fz2-C (green). Scale bar: 5 μm. (C) Schematic depicting the pathway of Wg binding to Fz2, resulting in postsynaptic nuclear localization of the receptor C-terminus. (D) Quantification of Fz2-C nuclear localization measured as Fz2-C puncta number in the nuclei, normalized to w¹¹¹⁸ genetic controls. Sample sizes are ≥7 animals and ≥14 muscles for each genotype. Significance is shown as *P<0.05 and **P<0.01.

Reduced Jeb ligand levels and dpERK signaling at dfmr1 null NMJs. (A) Representative NMJ images co-labeled with neuronal marker (HRP, red) and anti-Jeb (green) in control (w¹¹¹⁸) and dfmr1 null (dfmr1²) wandering third instar muscle 4. Right panels show synaptic boutons at higher magnification. Scale bars: 15 μm and 5 μm, respectively. (B) Quantification of Jeb intensity in two dfmr1 null alleles (dfmr1^50M, dfmr1²), normalized to genetic controls (w¹¹¹⁸). Sample sizes are ≥9 animals and ≥18 NMJs. (C) Representative images of individual muscle nuclei co-labeled with nuclear marker (PI, red) and anti-diphosphorylated extracellular signal regulated kinase (dpERK, green) in w¹¹¹⁸ and dfmr1^50M. Scale bar: 10 μm. (D) Quantification of dpERK intensity in two dfmr1 nulls (dfmr1^50M, dfmr1²), normalized to w¹¹¹⁸. Sample sizes ≥8 animals, ≥16 NMJs. Significance shown as **P<0.01 and ***P<0.001.

Restoration of dfmr1 null synaptic functional defect by HSPG reduction. (A) Representative excitatory junctional current (EJC) traces from the following six genotypes: genetic control (w¹¹¹⁸), w¹¹¹⁸ crossed to postsynaptic driver (24B-GAL4), homozygous dfmr1^50M null, 24B-GAL4 driving UAS-Dlp, heterozygous dlp^A187/+ recombined into the dfmr1 null background (dlp^A18, dfmr1^50M/dfmr1^50M) and double heterozygous dlp^A187/+ and sdc²³/+ in dfmr1 null background (sdc²³/+; dlp^A187, dfmr1^50M/dfmr1^50M). The nerve was stimulated in 1.0 mM external Ca²⁺ and TEVC records (−60 mV holding potential) made from muscle 6 in segment A3. Each trace is averaged from ten consecutive evoked EJC recordings. (B) Quantified mean EJC amplitudes (nA) for the six genotypes shown. Sample sizes are ≥8 animals and individual NMJ terminals. Statistical significance shown as *P<0.05, **P<0.01 and not significant (N.S.).

No detectable change in retrograde BMP signaling at dfmr1 null NMJs. (A) Representative NMJ images co-labeled with neuronal marker (HRP, red) and anti-Gbb (green) in control (w¹¹¹⁸) and dfmr1 null (dfmr1^50M) wandering third instar muscle 4. Right panels show synaptic boutons at higher magnification. Scale bars: 15 μm and 5 μm, respectively. (B) Quantification of Gbb intensity in two dfmr1 nulls (dfmr1^50M, dfmr1²), normalized to w¹¹¹⁸ genetic controls. Sample size is ≥8 animals and ≥16 NMJs for each genotype. (C) Representative images of motor neuron nuclei in the larval CNS co-labeled with nuclear marker (PI, red) and anti-pMAD (green) in w¹¹¹⁸ and dfmr1^50M. Arrows indicate motor neuron nuclei. Black/white images are shown to better highlight differences. Scale bar: 5 μm. (D) Quantification of pMAD intensity in two dfmr1 nulls (dfmr1^50M, dfmr1²), normalized to w¹¹¹⁸. Sample size is ≥15 animals and ≥30 NMJs for each genotype. Statistical significance is shown as N.S. (P>0.05).