Generation of neuromuscular specificity in Drosophila: novel mechanisms revealed by new technologies

Abstract

The Drosophila larval neuromuscular system is one of the best-characterized model systems for axon targeting. In each abdominal hemisegment, only 36 identified motor neurons form synaptic connections with just 30 target muscles in a highly specific and stereotypic manner. Studies in the 1990s identified several cell-surface and secreted proteins that are expressed in specific muscles and contribute to target specificity. Emerging evidence suggests that target selection is determined not only by attraction to the target cells but also by exclusion from non-target cells. Proteins with leucine-rich repeats (LRR proteins) appear to be a major molecular family of proteins responsible for the targeting. While the demonstrated roles of the target-derived cues point to active recognition by presynaptic motor neurons, postsynaptic muscles also reach out and recognize specific motor neurons by sending out cellular protrusions called myopodia. Simultaneous live imaging of myopodia and growth cones has revealed that local and mutual recognition at the tip of myopodia is critical for selective synapse formation. A large number of candidate target cues have been identified on a single muscle, suggesting that target specificity is determined by the partially redundant and combinatorial function of multiple cues. Analyses of the seemingly simple neuromuscular system in Drosophila have revealed an unexpected complexity in the mechanisms of axon targeting.

Keywords: Drosophila; motor neurons; muscles; neuromuscular junction; synapse specificity; target recognition.

Figure 1

Expression of target recognition molecules in specific muscles and motor neurons. (A) Expression of hemophilic cell adhesion molecules, Fas3, Capricious (Caps), and Connectin (Con) in specific synaptic partners. Fas3 is expressed in the RP3 motor neuron and its target muscles M6/7 (green). Caps is expressed in the RP5 motor neurons and its target M12 (orange). Con is expressed in external muscles and a group of motor neurons that innervate them (blue). (B) Expression of secreted factors, Netrin-B (NetB) and Wnt4 in specific muscles. NetB is expressed in M2, 6, and 7, whereas Wnt4 is expressed in M12 and 26. (C) Expression of Toll in specific muscles. Toll is expressed in subsets of ventral muscles including M6, 7, 13, 15, 16, 17, and 28 but not M12. AC and PC, anterior and posterior commissure. Projections of intersegmental (ISN, ISNb, and ISNd) and segmental (SNa and SNc) nerves are also shown.

Figure 2

Expression and function of muscle cues during the targeting of muscle 12 motor neurons. The expression of the muscle cues and their putative roles are shown in the left panel (A–C). Schematic diagram of their loss-of-function (LOF, A′–C′ and C″) and gain-of-function (GOF, A″–B″) phenotypes are shown on the right.

Figure 3

Transcriptional regulation of target specificity by repression of inhibitory cues. (A) Tey represses expression of Toll in M12 and thereby determines target specificity. (B) A model in which target specificity can be generated in a group of equivalent targets by transcriptional repression of a repulsive cue.

Figure 4

Partner recognition by myopodia. (A) Schematic diagram showing myopodia clustering and formation of nascent synaptic sites in wild type (above) and caps, trn double mutants (below). (B) Partner selection by myopodia (Left). While 54% of myopodial contacts with a partner motor neuron (magenta) are stabilized, none of the contacts with the non-partner motor neuron (blue) are stabilized (Right). Caps at the tip of myopodia may function in a bidirectional manner, providing target cues to and receiving signal from the presynaptic cells. (C) Caps accumulation at the tips of mypopodia. GC, growth cone (Top). Simultaneous live-imaging of Caps-GFP (green) and muscle membrane (magenta) reveals Caps localization at the tips of myopodia (arrows). Taken from Kohsaka and Nose (2009) Bottom. A diagram showing Caps localization.