The intricate pattern of motor output depends on the precise connectivity between motor neurons and muscles. As described above, there are 30 muscles in each abdominal hemi-segment of the body wall. These muscles are innervated in a highly stereotypic manner by approximately 40 motor neurons in the ventral nerve cord (VNC), which send their axons through one of the six branches of the peripheral nerves: intersegmental nerves (ISN, ISNb and ISNd), segmental nerves (SNa and SNc), and a transverse nerve (TN) (Fig. 2) (Keshishian et al. 2012; Landgraf & Thor 2003b). A majority of ISN, ISNb and ISNd motor neurons innervate muscles in the next posterior segment (thus the name “intersegmental”), whereas motor neurons of the segmental nerves (SNa and SNc) innervate muscles in the same segment. There is an anatomical correspondence between the choice of the peripheral branch and domains of the target muscles. ISN, ISNb and ISNd motor neurons innervate internal muscles in the dorsal (and dorso-lateral), ventro-lateral and ventral domains, respectively. SNa and SNc motor neurons innervate external muscles in the lateral and ventral domains, respectively (Fig. 2). Accordingly, the choice of the peripheral branch by motor neurons is crucial for matchmaking between motor neurons and muscles. Several transcription factors have been identified as regulating this process (reviewed in Landgraf & Thor 2003b). For example, the homeobox transcription factor Even-skipped (Eve) is necessary and sufficient for projection to the dorsal branch ISN (Landgraf et al. 2003a). Eve regulates the dorsal projection partly by suppressing the Unc-5 receptor (see below) (Labrador et al. 2008). Projection to ventral branches including ISNb and ISNd, is regulated by combined action of homobox proteins Nkx6 and Hb9 (Broihier & Skeath 1995). Even-skipped represses Nkx6 and Hb9, and vice versa, suggesting that mutual inhibition between these transcription factors determines the dorsal versus ventral projection of the motor neurons (Broihier & Skeath 1995). The ventrally projecting motor neurons are further specified by combined action of transcription factors including the LIM (lin-11/Isl-1/mec-3)-homeodomain-containing Islet and Lim3 and the POU (Pit-1/Oct-1/unc-86)-homeodomain containing Drifter, to make a choice between ISNb and ISNd (Thor et al. 2002; Certel & Thor 2001). Vertebrate orthologues of some of these transcription factors are also implicated in motor neuron specification, suggesting that the molecular mechanism of motor specification is evolutionarily conserved (Thor & Thomas 2004).
Several axon guidance molecules that directly regulate branch selection by motor neurons have been identified (reveiwed in Ruiz-Canada & Budnik 1997). Forward genetic screening conducted in the 1990s identified several mutants that show defects in motor axon projection, including beaten path and sidestep (Vactor et al. 2009; Sink et al. 1998). A recent study showed that Beaten path and Sidestep are a ligand-receptor pair for guiding axons to their targets (Siebert et al. 2002). Beaten path functions as a receptor expressed in motor neurons, whereas Sidestep functions as a ligand expressed on intermediate targets along the peripheral pathway (Siebert et al. 2002). Receptor tyrosine phosphatases DLAR and DPTP69D are required for ISNb and ISNd motor neurons to exit the common ISN pathway and extend towards the target muscles (Desai et al. 1950; Krueger et al. 2011). A heparan sulfate proteoglycans, Syndecan, has been identified as a ligand for DLAR (Fox & Zinn 2008; Johnson et al. 2010). Netrins and its receptors, Frazzled and Unc-5, also controls dorsal (ISN) versus ventral (ISNb) projection, by mediating attraction and repulsion to specific pathways (Labrador et al. 2008). Axon repulsion mediated by Semaphorin-1b (Sema-1b) is also required for the ISNb motor neurons to defasciculate from the ISN pathway and enter the target region (Winberg et al. 2011; Yu et al. 1998; Terman & Kolodkin 2007).
When motor neurons reach the target region by navigating through peripheral pathways, they finally find and synapse with their specific target muscle. Target recognition molecules, transmembrane or secreted proteins expressed on specific muscles, play critical roles in the choice of the partner muscle among a number of potential targets in the vicinity (reviewed in Nose 2010; Ruiz-Canada & Budnik 1997; Sanes & Yamagata 2006). Known target recognition molecules include attractive cues such as Fasciclin3 (Fas3) (Chiba et al. 2010), Connectin (Con) (Nose et al. 2010) and Capricious (Caps) (Shishido et al. 1996; Kurusu et al. 1996), as well as repulsive cues such as Wnt4 (Inaki et al. 2010), Toll (Rose et al. 2002; Inaki et al. 2011) and Sema-2a (previously termed SemaII) (Winberg et al. 2011). Motor neurons integrate information provided by multiple attractive and/or repulsive cues in determining which muscles to form synapse on (Winberg et al. 2011; Rose & Chiba 2000; Kurusu et al. 1996). When searching for the target muscles, presynaptic motor neurons extend numerous filopodia and contact a number of muscles in the target region including the non-target muscles. Postsynaptic muscles also extend numerous filopodia, called myopodia, which contact multiple motor neurons (Ritzenthaler et al. 2011; Kohsaka et al. 2009; Kohsaka & Nose 2001). Thus, matchmaking between motor neurons and target muscles appears to be a mutual recognition process in which both pre- and post-synaptic cells seek each other. A target recognition molecule, Caps, is localized at the tips of myopodia where initial contacts between motor neurons and muscles often occur (Kohsaka & Nose 2001). Thus, local and mutual interaction at the tips of myopodia might be crucial for target selection.
The basic pattern of neuromuscular connectivity is established by the end of embryogenesis and the pattern is maintained throughout larval life with minor changes in soma position, axon projection and dendrite morphology (Hoang & Chiba 2007; Kim et al. 1996). However, there is a dramatic increase in the size of the muscles during the larval period. To accommodate this change and maintain synaptic efficiency, the neuromuscular junctions expand, generating new branches and synaptic boutons (Schuster et al. 2000; Zito et al. 2007; Ruiz-Canada & Budnik 1997). This property makes this synapse an ideal model system to study the molecular mechanisms of synaptic growth and plasticity (reviewed in Ruiz-Canada & Budnik 1997; Collins & Diantonio 2004). Another important and well-characterized feature of the synapse is the homeostatic regulation that allows stable levels of synaptic activity (Davis 2011). The larval neuromuscular synaptic terminals are classified into larger type I endings, smaller type II endings and minor type III endings. Type I endings release glutamate, the main excitatory transmitter at this synapse and are further divided into type-I big (type-Ib) and type-I small (type-Is). Type-Ib motor neurons have bigger boutons, project to a single muscle, and are low-threshold, whereas type-Is motor neurons have smaller boutons, project to groups of muscles and are high-threshold (Choi et al. 2010; Schaefer et al. 2009). It has therefore been suggested that type-Ib and type-Is neurons are specialized for precise and powerful movements, respectively.
Dendrites and the myotopic map
Motor neurons receive information from the upstream central circuits connecting to their dendrites. Thus, the topology of motor neuron dendrites could be important in understanding the information flow in motor circuits. Comprehensive single cell analysis has been conducted to analyze the arrangement of motor neuron dendrites (Landgraf et al. 2011,1997). These studies revealed that dendrites of motor neurons form a map: dendrites of motor neurons innervating different muscle domains arborize in distinct regions in the neuropile along the anterior-posterior and medio-lateral axis (Fig. 2) (Landgraf et al. 1997; Kim et al. 1996; Mauss et al. 2005). Along the anterior-posterior axis, dendrites of motor neurons innervating the dorsal internal, dorso-lateral internal, ventral internal and external muscles occupy overlapping but distinct positions in the central nervous system (CNS) (Landgraf et al. 1997). A medio-lateral map was observed among internal muscles: dendrites of motor neurons targeted to ventral muscles extend more medially than those of more dorsally-targeted motor neurons (Kim et al. 1996; Mauss et al. 2005). Arrangement of the myotopic map likely reflects organization of the upstream neural circuits that control motor neurons.
What are the molecular and cellular mechanisms for drawing this map? Mauss et al. (2005) showed that the medio-lateral map is formed in part by the action of secreted factors expressed in the midline: the arrangement of dendrites is regulated by Netrin-Frazzled mediated attraction and Roundabout (Robo)-Slit mediated repulsion. Slit, a repellant secreted by the midline, regulates the positioning of dendrites at particular distances from the midline, and individual dendritic responses to Slit are mediated by cell-specific expression of Robo receptors. Similarly, Netrin, an attractant secreted by the midline, regulates the positioning of dendrites via the Frazzled receptor expressed on motor neurons. These findings suggest that the dendritic map can be generated in a genetically determined manner. There is evidence, however, that neural activity is also important for the growth of dendritic arbors (Tripodi et al. 2002). Blocking synaptic inputs to motor neurons induces overgrowth of the dendrites of motor neurons. Conversely, increasing the inputs induces a reduction in the arbors of the dendrites. Thus, the dendrites of motor neurons not only provide a spatially organized interface upon which upstream interneurons can connect, but also function as a variable device for adjusting motor output.