Identification of neural outgrowth genes using genome-wide RNAi.

Fly Genetics

The fly stock used to generating primary cell cultures for the full genome screen was w , elav C155 -GAL4 , UAS-mCD8-GFP/FM6;gcm-GAL4/CyO . The gcm-GAL4 line is a product of P-element exchange of the P[GAL4, w + ] with the P[lacZ, ry + ] element in the rA87 enhancer trap line [70] . Flies were raised in large populations and placed in population cages maintained at 25°C. For cell culture, embryos were collected on molasses plates streaked with paste made from autoclaved yeast mixed with water. For in vivo nervous system analysis, the Sec61α l(2)SH0190 [45] and Sec61α k04917 [71] alleles were analyzed. For unambiguous identification of homozygous mutant embryos, the Sec61α alleles were balanced over the CyO , wg en-lacZ chromosome.

Drosophila Cell Culture

Gastrula stage embryos ranging from 6 to 8 hours old were washed from molasses plates with 21°C water, and dechorionated in 50% household bleach in a Nytex-bottomed basket. Embryos were rinsed with sterile distilled water and transferred to Dounce homogenizers containing 100 mL Shields and Sang M3 media (Sigma), and homogenized using 10 pestle strokes. Homogenate was transferred to centrifuge tubes, and debris was pelleted twice with centrifugation at 40 g for 10 min and 5 min. Cells were pelleted by centrifugation at 380 g for 10 min. Cells were resuspended to 2×10 6 cells/mL using Shields and Sang M3 media (Sigma) supplemented with 10 U/mL penicillin, 10 ug/mL streptomycin, and 200 ng/mL insulin.

Full Genome RNAi Screen

At the Drosophila RNAi Screening Center at Harvard Medical School ( http://www.flyrnai.org ), dsRNAs were aliquoted in 5 µL volumes into plastic optical-bottomed, black 384-well plates (Corning), with each well containing 250 ng of dsRNAs ranging from approximately 200–500 bp. Specific details on dsRNA generation are previously reported [72] . 10 µL of primary cell preparation (at 2×10 6 cells/mL) were aliquoted to the dsRNA 384-well plates using a MultiDrop liquid dispenser, and incubated in a humidified chamber for 2 days at 18°C. Following serum-free incubation, cells were supplemented with 30 µL Shields and Sang M3 media (Gibco) containing 5% heat inactivated fetal bovine serum (JRH Biosciences), 10 U/mL penicillin, and 10 ug/mL streptomycin. Cells were incubated an additional 5 days at 18°C in serum-containing media. Following incubation, live, GFP expressing neurons and glia were scored visually, and 512×512 pixel images were collected with a Discovery1 automated widefield microscope (Universal Imaging). Raw images are available upon request. Control dsRNAs for Rho1, GFP, DIAP-1, and Neuroglian were included on each dsRNA plate. GFP dsRNA served effectively as a negative control, since GFP dsRNA wells were phenotypically indistinguishable from other negative controls such as lacZ dsRNA or water. The GFP dsRNA did not significantly knock down GFP signals in the cells due to the two very strong neuronal and glial promoters simultaneously driving GFP expression, in addition to protein perdurance effects. Except for Rho1, GFP, DIAP-1, and Neuroglian controls on each plate, all wells were scored blind. Importantly, dsRNAs for Rho1, DIAP-1, and Neuroglian were also represented in the dsRNA collection, and were detected as hits when scored blind. The primary screen was carried out on 1–2 independent replicates. For secondary screening, dsRNAs were resysnthesized as reported previously [73] . The secondary screen dsRNAs were aliquoted to 384-well plates in 2 different orientations to avoid plate location artifacts. The secondary validation screens were carried out on 12 independent replicates. For each gene 2 to 3 additional non-overlapping dsRNA amplicons were generated that had no 17–19 bp matches to other genes. Details of the dsRNAs used are publicly available at the DRSC website: www.flyrnai.org . Digital Image Analysis Image Feature Extraction of Cell Clusters From the automated widefield microscopy images, components of the cell phenotypes were quantified. First, neuron cell clusters sizes were determined using the Otsu method (Otsu, 1979), which chooses a global threshold to minimize the intraclass variance of the background and foreground pixels, to convert a raw intensity image to a binary image. The foreground was divided into three categories: small dots (area≤16 pixels), small cell clusters (16 pixels