MARCM analysis demonstrates requirement of extra- and intracellular domains of Nrg180 for synapse stability.

<p>(A) Overview of the genomic locus of <i>nrg</i>. The Pacman construct spans 92 kb including the endogenous enhancer elements up- and downstream of <i>nrg</i>. The Nrg167- and Nrg180-specific exons and the relevant amino acid sequences are depicted. The position of the common Ig-domains 3 and 4 is indicated. The isoform-specific FIGQY sequences are highlighted in red and the PDZ protein-binding motif of Nrg180 isoform is underlined. (B) A <i>nrg¹⁴</i> MARCM clone rescued by a wild-type <i>nrg</i> Pacman construct. The motoneuron clone was marked by the expression of mCD8–GFP (green). Synaptic vesicles (DvGlut, red) were found opposite postsynaptic Dlg (blue), indicating a stable NMJ. (C) A <i>nrg¹⁴</i> MARCM clone showing a synaptic retraction. Only fragmented remnants of the membrane GFP marker were still present at a nerve terminal that almost completely lacked the presynaptic active zone marker Brp (red). Postsynaptic glutamate receptor clusters were still present. In addition, the axonal membrane prior to the NMJ was also fragmented. (D) A <i>nrg¹⁴</i> MARCM clone expressing only Nrg180 lacking the FIGQY motif. The mutant (GFP-positive) NMJ was retracted while a neighboring control NMJ (asterisk) remained stable. (E) Composite image overview of an <i>nrg¹⁴</i> MARCM clone expressing a mutated form of Nrg180 lacking the extracellular Ig3–4 domains. Three areas are shown at larger magnification in 1–3. (E¹) At the NMJ no presynaptic vesicles were present opposite postsynaptic Dlg. The Dlg staining was no longer interconnected and only remnants of the presynaptic membrane marker mCD8–GFP were visible, indicating a complete elimination. (E²) Approximately 150 µm proximal from the NMJ, the axon ended in a “bulb-like” structure. Between the “bulb-like” axon ending and the NMJ, only punctate staining of the membrane marker was visible. (E³) At a significant distance from the “bulb,” a large axonal swelling was visible that contained aggregates of the synaptic vesicle marker DvGlut. (F) Quantification of mCD8-marked axons that were not connected to target muscles in the indicated genetic background. The <i>nrg¹⁴</i> mutant phenotype was significantly rescued by the presence of a wild-type <i>nrg</i> Pacman construct, a <i>nrg</i> construct lacking the FIGQY domain of Nrg167, and only partially rescued by P[<i>nrg180^ΔFIGQY</i>] and not rescued by P[<i>nrg180^ΔIg3–4</i>]. (G) Analysis of muscle innervation pattern of motoneuron MARCM clones that were connected to postsynaptic muscles. In all genotypes we observed normal innervation patterns for all four major classes of motoneurons. Scale bar in (B) corresponds to (B-D), 10 µm. Scale bar in (E), 20 µm. Error bars represent SEM.