6 ± 0.8, p < 0.0001 paired Student's t test; HgfpH 3.6 ± 0.7, p < 0.05 paired Student's t test; Figure 2C). Similar results were observed when reporter expression
was compared in all DD and VD neurons (DD/VD fluorescence ratios: HgfpC 5.6 ± 0.5, p < 0.0001 paired Student's t test; HgfpH 2.6 ± 0.4, p < 0.005 paired Student's t test; Figures 2A and 2B and Figures S2B and S2D). learn more These results indicate that the hbl-1 promoter is expressed at significantly higher levels in DD neurons than in VD neurons. The decreased hbl-1 reporter expression in VD neurons could result from UNC-55 mediated repression of the hbl-1 promoter. To test this possibility, we analyzed expression of the HgfpC reporter in unc-55 mutants. HgfpC expression in VD neurons was significantly increased in unc-55 mutants (197% wild-type levels, p < 0.001 Student's t test), indicating increased transcription of the hbl-1 promoter in unc-55 mutant VD neurons ( Figure 2D and Figures S2B–S2D). The magnitude of the increased HgfpC expression differed in individual VD neurons. For VD10, HgfpC
expression in unc-55 mutants rose to the same level observed in DD5 neurons ( Figure 2D); however, in most cases, HgfpC expression in unc-55 mutant VD neurons remained significantly lower than that observed in DD neurons (DD/VD fluorescence ratio in unc-55: HgfpC 2.3 ± 0.4, p < 0.001 Student's t test; Figures S2C Y-27632 and S2D). By contrast, HgfpC expression in DDs did not increase in unc-55 mutants and instead was modestly decreased ( Figure 2D and Figure S2D). This is unlikely to be a direct effect of UNC-55 on the hbl-1 promoter because unc-55 is not expressed in DD neurons
( Zhou and Walthall, 1998). Taken together, these data support the idea that UNC-55 inhibits expression of the hbl-1 promoter in VD neurons and that hbl-1 expression in D neurons is likely regulated by additional factors beyond UNC-55. In Drosophila, the UNC-55 ortholog (Sevenup) represses Hunchback (Hb) transcription ( Kanai et al., 2005 and Mettler et al., 2006). As in Drosophila, the C. elegans hbl-1 promoter contains four predicted UNC-55 binding sites, suggesting the hbl-1 could be a direct target for UNC-55 repression. To test this idea, we mutated the UNC-55 binding sites in the almost hbl-1 promoter, and assayed its expression pattern. The mutant hbl-1 promoter (HmutgfpC) had a significantly reduced DD5/VD10 expression ratio (HgfpC 6.6 ± 0.8; HmutgfpC 2.7 ± 0.3, p < 0.0001 Student’s t test) ( Figure 2E), which was not significantly different from the ratio observed for the wild-type reporter (HgfpC) in unc-55 mutants (1.8 ± 0.3, p = 0.17, Student’s t test) ( Figure 2F). Thus, the UNC-55 binding sites are required for differential expression of the hbl-1 promoter in VD and DD neurons. If UNC-55 repression of hbl-1 prevents VD remodeling, we would expect that mutations reducing hbl-1 activity would diminish ectopic remodeling of VD synapses in unc-55 mutants.