Note, however, that we were able to assess the requirement for homophilic binding in da neurons from the knockin alleles by using iMARCM, as reported in the previous section, because expression from the endogenous locus does not rely on GAL4. Unfortunately, iMARCM does not facilitate expression of two chimeric

isoforms encoded at the endogenous locus in the same cell. Thus, to test for cell-autonomous rescue of self-avoidance by complementary isoforms, we used MARCM analysis in MB neurons. cDNAs that encode Dscam1 isoforms, both wild-type and chimeras, selleck chemicals were placed under the control of the upstream activating sequence (UAS) enhancer and were inserted into a defined genomic position through phiC31 site-specific recombination (Groth et al., 2004). Different isoforms were expressed at similar levels as assessed by western blots of extracts that were prepared from embryos in which UAS expression was driven by a panneuronal GAL4 transgene (data not shown). Consistent with iMARCM experiments (Figures 2 and S5), expression of two copies of any of the four chimeras

only provided weak self-avoidance activity in Dscam1null MB neurons ( Figure 3A). Expression of either pair of complementary isoforms (i.e., a single copy of each UAS transgene inserted into the same site on two homologous chromosomes), however, rescued the branch segregation defect to a similar extent to the wild-type transgenes ( Figure 3A). Mephenoxalone Thus, Dscam1 acts in a cell-autonomous fashion through direct binding of A-1210477 order complementary protein domains on sister neurites of the same cell ( Figure 3B). These data establish that binding between matching isoforms is essential for Dscam1

function in vivo. If Dscam1 isoform-specific recognition does, indeed, play an instructive role in self-recognition, then expressing two different, yet complementary, isoforms on neurites of different cells should also elicit a repulsive response between them. To test this, we expressed chimeric isoforms alone or in combination with a complementary isoform in da neurons and explored the dendritic arbor patterns elaborated by class III (v’pda) neurons relative to the dendrites of class I (vpda) neurons (Figure 4). In wild-type animals, the class I dendritic arbor pattern is established first (Soba et al., 2007). Subsequently, the class III neurons elaborate dendrites, which overlap with the dendrites of class I neurons (Hughes et al., 2007, Matthews et al., 2007 and Soba et al., 2007) (Figures 4A and 4E). Expression of a wild-type Dscam1 isoform in both cells induced repulsion and, as a result, there were few overlaps between their dendrites (Hughes et al., 2007, Matthews et al., 2007 and Soba et al., 2007) (Figures 4B and 4E). Only weak ectopic repulsion was seen in response to expression of each Dscam1 chimera (Figures 4C and 4E).