UNC-40/DCC induces synapse formation in response to a local source of UNC-6/netrin.

The development of neural circuit requires appropriate axon guidance and synapse formation decisions. How are these processes coordinated in vivo is not well understood. We are attempting to understand the wiring of the nerve ring by initially focusing on the C. elegans thermotaxis circuit. This circuit includes one sensory neuron, AFD, two first-order interneurons AIY and AIZ, and a second-order interneuron RIA, which receives inputs from AIY and AIZ to mediate thermotaxis behavior (Mori, 1999).

We found that the connectivity between AIY and RIA neurons is orchestrated by a pair of glial cells that express UNC-6/Netrin. The processes of AIY and RIA meet at a specific coordinate within the nerve ring (dashed box in Fig. 4-1), where AIY synapses onto RIA (Fig.4-1 A , B, D). A pair of astrocyte-like glial cells send endfeet adjacent to the AIY-RIA synapses (Fig. 4-1C). In the postsynaptic neuron RIA, the Netrin receptor UNC-40/DCC plays a conventional guidance role directing the outgrowth of the RIA process ventrally towards the glia endfeet. In the presynaptic neuron AIY, UNC-40/DCC plays an unexpected role: it cell-autonomously promotes assembly of presynaptic terminals in the immediate vicinity of the glial cell endfeet (Fig. 4-1B). In unc-40 or unc-6 mutants, RIA fails to contact AIY and the AIY presynaptic specializations form at abnormal locations. These results indicate that Netrin can be utilized both for axon guidance and local synaptic differentiation, and suggest that glial cells can function as guideposts during the assembly of neural circuits in vivo.

Interestingly, both AIY and RIA neurons use the same extracellular cue, UNC-6/Netrin, to produce different developmental outputs, axon extension and synapse formation. We hypothesize that the distinct cell-autonomous actions of UNC-40 are generated by different downstream effectors. We are currently using three approaches to understand how UNC-40 can regulate axon guidance and presynapse formation. First, we are performing structure-function analysis attempting to identify the cytoplasmic region of UNC-40 receptor that is required for AIY synapse patterning. We will compare this region with the known UNC-40/DCC domain required for axon guidance. Second, we are examining the known UNC-40/DCC downstream signaling components for potential roles in synapse formation. Third, we are cloning several mutants that show similar AIY synapse patterning phenotype as unc-40, and that were isolated in the same initial screen. Through these approaches, we hope to gain insights into how different downstream signaling pathways are coupled to UNC-40 to generate diverse cellular outcomes such as axon guidance and presynaptic assembly.

Fig. 4-1 Characterization of AIY-RIA synapses. (A) trajectory of AIY(red) and RIA(green). (B, D) the AIY presynaptic vesicles (red) and RIA postsynaptic receptors (green) are localized at discrete subcellular regions. (C) Glia-like sheath cell (purple) endfeet physically contact the synaptic region between AIY and RIA.