Neural circuits of complex brains are frequently organized into parallel layers (laminae), with distinct populations of afferents innervating specific layers. Such layer specificity is widely observed in vertebrate and invertebrate brains, and it is likely a major determinant of synaptic specificity in the central nervous system. Our research group studies the development of layer-specific connections using Drosophila visual system as a model. The Drosophila compound eye consists of approximately 800 ommatidia, each containing three types of photoreceptor neurons (R1-6, R7, and R8). The R1-6 neurons respond to green light and connect to the first optic ganglion (the lamina), whereas the R7 and R8 are sensitive to ultraviolet and blue lights, respectively, and connect to the second optic ganglion (the medulla). The medulla is subdivided into ten layers (M1-10). The information from these three types of photoreceptor neurons is directed to distinct medullar layers: the R7 and R8 directly connect to the M6 and M3 layers, respectively, while the laminal neurons (L1-5) relay R1-6 input to multiple medullar layers. The establishment of layer-specific connections by different afferents in the medulla is critical for various visual functions, including color vision. In a genetic screen based on innate color-discrimination behavior, we have previously identified 5 loci that affect R7 connectivity. These are three molecularly characterized loci, N-cadherin, receptor phosphatase LAR, and milton, as well as two novel loci. These mutants provide molecular handles for dissecting the mechanisms of R7 layer-selection. To gain insight into the developmental mechanisms governing the formation of layer-specific connectivity within the medulla, we examined the innervation of medulla by R7 and R8 afferents at various developmental stages. We found that R7 and R8 layer-specific targeting occurs in two distinct stages. During the first stage, R7, R8, and LN axons sequentially target to their temporary layers. At the second stage, the R7 and R8 growth cones regain motility and project synchronously to their destined layers. Developmental analyses revealed that N-cadherin and LAR are required during the first stage of R7 layer-selection, while the other loci likely affect the second stage. The two-step target selection has been observed in the vertebrate hippocampus: during embryonic development, entorhinal axons and commissural and associational axons form transient synapses with Cajal-Retzius cells and GABAergic interneurons, respectively, before they synapse onto their postnatal targets, the pyramidal neurons. The two-stage R7 layer-selection might serve to coordinate afferent innervation with target development, as in hippocampus. Alternatively, it might function to reduce the number of potential targets among which R7 growth cones must choose. Using the mutations that delete different afferent subsets or alter R7 connectivity, we defined the cellular mechanism of R7 layer-selection. The genetic cell-ablation results suggest that R8 and R7 axons target to their temporary layers independently. In addition, the wild-type R8 axons target correctly when the neighboring Ncad or LAR mutant R7s mistarget to the R8-recipient layer. Conversely, the removal of Ncad in single R8s disrupts R8 targeting without affecting the targeting of the neighboring R7s. Thus, in contrast to the R1-6 target selection where afferent-afferent interactions play a key role, medullar layer-selection by R8 and R7 afferents likely involve primarily afferent-target interactions. Developmental analyses of single Ncad mutant R7s revealed that Ncad is required for R7 axons to reach and to remain in the R7-temporary layer. On the basis of its homophilic activity and mutant phenotypes, we propose that Ncad mediates the interaction between the R7 growth cones and the R7-temporary layer. The comparison of the first and second stagessss of R7 layer-selection derives further insight into the underlying mechanism. First, targeting to the R7-temporary layer at the first stage appears to be critical for the R7 axons to reach their final destination. Ncad or LAR mutant R7 axons that mistarget to the R8-temporary layer at the first stage, later proceed to terminate incorrectly as well at the R8-recipient layer. Second, in contrast to the initial target selection, which occurs immediately after the axonal projection, all R7 and R8 axons enter the second targeting stage at approximately the same time regardless of when they arrive at the medulla. We speculate that a global signal is responsible for triggering the initiation of the second stage. We are currently investigating the loci that might affect the second stage of R7 target selection to determine the nature of this signal.