Fig. 6. Excessive postsynaptic activity modulates axon growth and synapse formation. Confocal analysis of 17 hpf wild-type and nic1^{twister dbn12} mutant embryos, stained with antibody F59 specific for slow muscle fibers (A-C), or double stained with motoneuron-specific antibodies (D,G,J,M-O), and with AlexaFluor 594-conjugated -BTX to visualized clustered AChRs (E,H,K,M-O) at 17 hpf (A-L) or 26 hpf (M-O; all lateral views). (A,B) In 17 hpf wild-type and nic1^{twister dbn12} heterozygous mutant embryos, muscle fibers are thin and elongated but few display striations. (C) In nic1^{twister dbn12} homozygous mutant embryos, muscle fibers are less elongated with no visible striations. In homozygous mutant embryos, somites are compressed along the anterior-posterior axis and expanded along the dorso-ventral axis. (D-F) In 17 hpf wild-type embryos, motor axons have reached or just extended past the choice point (the level is indicated by white bars). Note the elaborate and fan-like morphology of the axonal tip, characteristic of advancing growth cones (white double arrowhead). As motor axons pioneer into the somites, clustered AChRs emerge and colocalize along the extending axon, reminiscent of en passant synaptic contacts. These dense AChR clusters decorate parts of the axon, except for the presumptive growth cone (white arrow) which precedes the distal limit of clustered AChR (white arrowhead). (G-I) In heterozygous embryos, most motor axons extend normally and their presumptive growth cones display a wild-type-like morphology (white arrow). However, a significant fraction of heterozygous motor axons stall before reaching the choice point, and presumptive growth cones appear smaller and less elaborate (red arrow). On those axons, dense AChRs clusters are localized distal to presumptive growth cones (white arrow). Note that the morphology of these aneural AChR clusters is indistinguishable from those that co-localize with the axon. (J-L) In nic1^{twister dbn12} homozygous mutant embryos, many motor axons (red arrows) are stalled before the choice point. Growth cones are less elaborate and appear collapsed (white double arrowhead), and AChR clusters are smaller and scattered throughout the somite (white arrowheads). (M) In 26 hpf wild-type embryos, AChR clusters are restricted along the lengths of the ventral and dorsal motor axons (yellow arrows) and along the somite boundaries (yellow arrowhead). (N) In nic1^{twister dbn12} heterozygous embryos, AChR clusters co-localize along the lengths of the ventral and dorsal axons (yellow arrows) and along the somite boundary (yellow arrowhead). AChR clusters are also detected along aberrant branches (blue arrowhead). (O) In 26-hpf nic1^{twister dbn12} homozygous mutants, smaller and fewer AChR cluster co-localize with axonal branches (blue arrowhead). Somite boundary localization of clustered AChRs is strongly reduced. Scale bar: 50 µm.