, 2006) in which R7 axons fail to terminate at the M6 layer, but rather target the M3 layer. However, the observed “gaps” may also be caused by a loss of R7 cells. We therefore stained third-instar larval eye discs with anti-Elav ( Figures S2A and S2B) and 24B10 antibodies ( Figures S2C and S2D) to reveal the differentiation of neurons and R cells. To determine if specific PR cells were properly identified, we also labeled R4 cells

with mΔGFP ( Cooper and selleck compound Bray, 1999; Figures S2E and S2F), R7 cells with 181Gal4 GFP ( Lee et al., 2001; Figures S2C and S2D), and R8 cells with anti-Senseless ( Nolo et al., 2000; Figures S2A and S2B). We observed no difference in staining pattern for any of the markers between 3L6 mutant and wild-type cells, indicating that the differentiation of PR cells is not affected in the mutants. We then analyzed retinal thick sections of adult flies and did not observe loss of R7 cells in the 3L6 mutants ( Figures S2G and S2H), although a rare ommatidium has an abnormal morphology. In contrast, labeling of the R7 terminals with UAS-Synaptotagmin GFP (SytGFP) drived by Pan-R7-Gal4 ( Ratnakumar and Desplan, 2004a), showed that about 20% (19.7% ± 3%, n = 268) of all R7 cell terminals fail to reach their target layer M6 but target the M3 layer ( Figures 2A, 2B, 2A′, and 2B′) click here in the adult brains of eyFLP; 3L61 mutants. Note that

the targeting defect is an underestimate (see Figure 5B) since we did not label the mutant clones and the 3L61 mutant clones are small because of a growth disadvantage with respect to heterozygous cells. Finally, we assessed the projection pattern of R8 cells by labeling them with Rh6-GFP ( Ratnakumar and Desplan, 2003). 3L6 mutant animals do not exhibit any obvious R8 targeting defects ( Figures 2C, 2D, 2C′, and 2D′). Since the phenotypes associated with loss of 3L6 are specific and interesting, we performed meiotic recombination mapping using P

elements ( Zhai et al., 2003). Rough mapping placed 3L6 in the 77A4–79F4 cytological interval. Deficiency mapping mapped 3L6 to 79C2–80A4. why As recombination frequencies are extremely low in this interval, we generated four small overlapping deficiency using FRT bearing P elements and PiggyBac insertions ( Parks et al., 2004 and Thibault et al., 2004; Figure 3A). Complementation tests narrowed the putative gene down to six genes and sequencing revealed two premature stop codons in CG9063 at amino acid (aa) 380 (3L61) and 1196 (3L62) ( Figure 3A). Since 3L61 has an early stop codon, it is probably a null or a strong hypomorphic allele, whereas the 3L62 allele contains a late stop codon, indicating that it may be a hypomorphic allele of CG9063. These data are in agreement with all the phenotypic data. Note that the 3L62 allele causes a weaker ERG and R7 targeting defect than the 3L61 allele (Figures 1A, 1D, 7E, 7F, and 7M).