DEV01661 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Axons originating from L1-L5 neurons project into a layer between the R7- and R8 temporary layers during 17-35% APF. Single L1-L5 neurons were labeled using the MARCM system and UAS-mCD8-GFP, and visualized with anti-GFP antibodies. At this stage, all L1-L5 neurons (L1-5) express Elav while only L4 and L5 express Bsh. The GFP-labeled L1-L5 neurons were identified by the positions of their cell bodies in the L1-L5 columns. (A,A¢) L1-L5 projections were assessed at 17% APF. A single L2 neuron was labeled and its axon was traced to the layer (arrow) between R7 and R8 growth cones. (B,B¢,B¢¢) L1-L5 projections were assessed at 35% APF. Three L2 neurons were labeled by the MARCM system. Their growth cones (arrows) terminated at the layer between the R7- and R8 temporary layers. In this specimen, an R7 growth cone was also labeled (arrowhead). (A¢,B¢,B¢¢) High magnification views of A and B, respectively. Scale bars: in A,B, 30 mm; in A¢,B¢,15 mm.

• Supplemental Figure S2 -

  Fig. S2. The development of medulla neuropil during the second layer‑selection stage. The development of medulla neuropil was assessed using Apterous‑Gal4 UAS‑mCD8GFP marker (green), which labeled medulla neurons as well as L4, lobula and lobula plate neurons. R-cell axons were labeled with Mab24B10 (red). A,A¢,A¢¢; B,B¢,B¢¢; C,C¢,C¢¢; D,D¢,D¢¢; and E,E¢,E¢¢ are at 40%, 50%, 60%, and 70% APF and the adult stage, respectively. The presumptive R7 temporary and R7 receipient layers are indicated by dotted and broken lines, respectively (A¢¢-E¢¢). Medulla layers M3-6 are as indicated. As the medulla neuropil develops, its size and complexity increase, and its columnar structure becomes prominent. (A¢¢-E¢¢) The red channel was removed from A¢-E¢ to facilitate the visualization of the medulla neuropil. A¢,A¢¢; B¢,B¢¢; C¢,C¢¢; D¢,D¢¢; and E¢,E¢¢ are high magnification views of A, B, C, D and E, respectively. ed, eye disc; la, lamina; me, medulla: lo, lobula; MN, medulla neurons. Scale bars: in A, 30 mm for A-E; in A¢, 10 mm for A¢-E¢.

• Supplemental Figure S3 -

  Fig. S3. Removing Ncad in R8s or small patches of medulla neurons does not affect R7 layer selection. R7 targeting was assessed at 17% APF (A-A¢) and at 40% APF (B-D). Ncad mutant R8 (A,A¢) or medulla neurons (B-D) were generated using hs-Flp-mediated mitotic recombination (see Materials and methods), and labeled using the MARCM system and mCD8-GFP. R7 and R8 axons were visualized with Mab24B10 (red). (A,A¢) Ncad mutant R8 growth cones (arrows) retracted from the R8 temporary layer. Nevertheless, the corresponding R7 growth cones (double arrows) target correctly to the R7 temporary layer. (B-D) The layer-selection of R7 growth cones was not affected by the small patches of Ncad mutant medulla neurons. Note that medulla neurons are highly heterogeneous and exhibit various dendritic arbors (arrows) in different layers. (A¢,B¢) High magnification views of (A) and (B), respectively. Scale bars: in A,B, 30 mm;  in A¢, B¢,C-D, 10 mm.

• Supplemental Figure S4 -

  Fig. S4. Sequence alignment of Ncad amino-acid sequences encoded by alternative exons. The amino-acid sequences of the variable region of Ncad protein from four insect species, Drosophila melanogaster (Dm), Drosophila pseudoobscura (Dp), Anopheles gambiae (Ag), and Apis mellifera (Am) are shown. Exons corresponding to Drosophila exons 13b and 18b have not been identified in Apis mellifera. The amino-acid residues that are conserved in all isoforms are colored red. Those conserved in 7a, 13a, and 18a are colored blue, and those in 7b, 13b, and 18b are colored green. The predicted secondary structure of cadherin, shown above the sequences, is modeled on the basis of known vertebrate cadherin structures (Pertz et al., 1999; Tamura et al., 1998). The cadherin and EGF-CA residues that likely mediate calcium binding are indicated by red asterisks. The conserved cysteine residues that might form disulfate bonds in the EGF‑CA domains are marked with a brown letter C.

• Supplemental Figure S5 -

  Fig. S5. Over- or mis-expressing one Ncad isoform in the wild-type background, results in transient R7 targeting defects. The Ncad isoform 7b-13a-18a (A,A¢) or 7b-13a-18b (B,B¢) was expressed in R7 cells at 17% APF using PM181-Gal4 UAS-Ncad, and the R7 axons were labeled with mCD8‑GFP. In the younger part of the medulla, the R7 growth cones expressing Ncad isoforms are disorganized and often extend filapodia deeper into the medulla. However, these defects are greatly reduced in the older part of the medulla where the PM181 expression in older R7 growth cones is at a low level (angular bracket). The presumptive R7- and R8 temporary layers are indicated by dotted lines in A¢,B¢. (A¢,A¢¢,B¢,B¢¢) High magnification views of A,B, respectively. Scale bars: in A, 30 mm for A,B; in A¢, 10 mm for A¢,B¢.

• Supplemental Figure S6 -

  Fig. S6. Western blot analysis of the Ncad isoform expression in S2 cells. S2 cell expressing GFP (lane 1), Ncad7b-13a-18a (lane 2), Ncad7a-13a-18a (lane 3), Ncad7b-13b-18a (lane 4), and Ncad7b-13a-18b (lane 5). Rat monoclonal antibodies against the intracellular domain of the Ncad protein were used to quantify the Ncad expression. Double asterisks and single asterisks indicate the unprocessed form and the processed C-terminal fragment of Ncad proteins, respectively.