Figure 4. Regulation of Retrograde Signaling by Glia-Derived Mav.

(A) Real-time PCR of larval muscle RNA, showing that dad and gbb transcripts are significantly decreased in muscle when Mav is downregulated in NMJ glia (n = 3).

(B) Normalized P-Mad intensity in motor neuron nuclei in the indicated genotypes, showing that Mav downregulation in glia results in a reduction in P-Mad levels in neurons (from left to right, n = 12, 11, 10, 8, 9).

(C) Real-time PCR of larval brain RNA, showing that Trio transcript is reduced in brain when Mav is downregulated in glia (n = 3).

(D–F) View of larval ventral ganglia shown at (D1–F1) low and (D2–F2) high magnification in preparations double labeled with anti-Elav and anti-P-Mad antibodies in (D) wild-type and larvae expressing (E) Mad-RNAi in neurons or (F) Mav-RNAi in peripheral glia.

(G) Number of boutons on muscle 4 (segment A3) in the indicated genotypes, demonstrating that removal of a single copy of gbb suppresses the effect of overexpressing Mav-GFP in glia (from left to right, n = 11, 12, 12, 13, 18, 12).

(H) Normalized number of boutons on muscles 6 and 7 in controls (UAS-Punt-RNAi/+) or in larvae expressing the Punt-RNAi constructs using C57-Gal4 (Punt-RNAi-muscle). Samples were normalized to their respective controls to allow direct comparisons (from left to right, n = 12, 13, 10, 12).

(I) Normalized P-Mad intensity in motor neuron nuclei in control and Punt-RNAi-1-muscle samples, showing reduction of P-Mad signal when Punt is downregulated in muscles (from left to right, n = 35, 34).

(J) Model of glia-derived Mav signaling. Mav is secreted by glia and binds to the type II receptor Punt, which together with an unknown type I TGF-β receptor phosphorylates postsynaptically localized Mad. P-Mad dimerizes with the co-Smad Medea (Med) and translocates into the muscle nucleus, where it activates Gbb transcription. Muscle Gbb is released by muscles and retrogradely activates Wit and Tkv or Sax receptors in presynaptic terminals. This leads to presynaptic phosphorylation of Mad, which in a complex with Med translocates into motor neuron nuclei and activates Trio transcription. Trio is transported to presynaptic endings, where it regulates synaptic bouton growth.

Error bars represent mean ± SEM (***p < 0.001, **p ≤ 0.01, *p ≤ 0.05). Scale bar represents 10 μm for (D2)–(F2) and 25 μm for (D1)–(F1).