Daughterless, the Drosophila orthologue of TCF4, is required for associative learning and maintenance of the synaptic proteome

ABSTRACT Mammalian transcription factor 4 (TCF4) has been linked to schizophrenia and intellectual disabilities, such as Pitt–Hopkins syndrome (PTHS). Here, we show that similarly to mammalian TCF4, fruit fly orthologue Daughterless (Da) is expressed widely in the Drosophila brain. Furthermore, silencing of da, using several central nervous system-specific Gal4 driver lines, impairs appetitive associative learning of the larvae and leads to decreased levels of the synaptic proteins Synapsin (Syn) and Discs large 1 (Dlg1), suggesting the involvement of Da in memory formation. Here, we demonstrate that Syn and dlg1 are direct target genes of Da in adult Drosophila heads, as Da binds to the regulatory regions of these genes and the modulation of Da levels alter the levels of Syn and dlg1 mRNA. Silencing of da also affects negative geotaxis of the adult flies, suggesting the impairment of locomotor function. Overall, our findings suggest that Da regulates Drosophila larval memory and adult negative geotaxis, possibly via its synaptic target genes Syn and dlg1. These behavioural phenotypes can be further used as a PTHS model to screen for therapeutics. This article has an associated First Person interview with the first author of the paper.

GMR12B08>mCD8-GFP.Additional Da protein forms appear when Da is overexpressed using ubiquitous strong driver da G32 -Gal4 but are not detected using nervous system specific GMR12B08-Gal4.

1Figure S1 .
Figure S1.Da overexpression in embryos using ubiquitous driver da G32 -Gal4 reveals additional signals using anti-Da antibody.Western blot analysis of embryos with anti-Da antibody dam109-10, β-tubulin (β-tub) was used as loading control.Control embryos are

Figure S2 .
Figure S2.Silencing of da in larval brain reduces Da levels.A -Western blot analysis with anti-FLAG antibody of dissected third instar larval brains.Numbers on the left indicate molecular weight in kDa, 3xFLAG-da -larval brains where Da is tagged with 3xFLAG epitope, Control -3xFLAG-da;GMR12B08>Dcr2 larval brains, da RNAi -3xFLAGda;GMR12B08>Dcr2,da RNAi larval brains, wt -white 1118 larval brains for control lacking the 3xFLAG tag.B -upon silencing of da the Da expression levels were reduced about 35% compared to 3xFLAG-da and about 25% compared to 3xFLAG-da;GMR12B08>Dcr2 larval brains.Shown are the results of densitometric analysis of Western blot, 3xFLAG-Da signals were normalized using β-tubulin signals.The mean results from three independent Western blots are shown, blue bars represent mean intensity of 80 kDa protein signal and orange bars represent mean intensity of 65 kDa signal.Error bars show standard error of the mean (s.e.m).Statistical significance is shown with asterisks between the groups connected with lines.*P<0.05,**P<0.01,One Way ANOVA with post-hoc Bonferroni test.

Figure S3 .
Figure S3.Overexpressing da has moderate positive effect on memory deficit caused by silencing of da.UAS-Dcr2;UAS-da RNAi ;12B08-Gal4/+ -da RNAi -larve zero PI while UAS-Dcr2;UAS-da RNAi ;12B08-Gal4,UAS-da/+ -da RNAi ,da -larve have non-zero PI.PI-s are visualized using box-whisker plots, which show the median, the 25% -75% quantiles (boxes), and the minimum to maximum (whiskers).To determine PI difference compared to zero inside one genotype one-sample sign test was used (asterisks indicated over the boxes) and between the groups Mann-Whitney U-test was used in statistical analysis.

Figure S6 .
Figure S6.Da is expressed widely in the adult Drosophila brain including the central brain thoracic ganglion.A-A'' -Da is expressed in many cells marked by 30Y-Gal4.B-B'' Da is expressed in some cells marked by OK107-Gal4.C-C'' -Da is expressed in a few cells marked by R12B08-Gal4.D-D'' -Da is expressed in a few cells marked by 201Y-Gal4.Driver expression is visualized with nls-GFP (A', B', C' and D') and 3xFLAG-Da is magenta (A'', B'', C'' and D'').Scale bar represents 70 μm on A and 30 μm on D.

Figure S7 .
Figure S7.Mutations in bHLH domain of Daughterless and TCF4 abolish their transactivational capability.Cultured rat primary neurons were with constructs encoding empty vector, wild type Da, R580W mutated Da (Da mut ), TCF4A, A587P mutated TCF4A (TCF4A mut ), TCF4B or A587P mutated TCF4B (TCF4B mut ), firefly luciferase construct carrying 12 µE5 boxes with a TK promoter, and Renilla luciferase construct with mouse PGK promoter for normalization.Luciferase activities were measured and data are presented relative to the luciferase signals obtained from cells transfected with wild type daencoding construct and treated with 0.1% DMSO for control.Mean results from three independent transfection experiments performed in duplicates are shown.Error bars show standard error of the mean (s.e.m).Statistical significance is shown compared to cells expressing respective non-mutated effector protein and treated similarly.*P<0.05,One Way ANOVA with post-hoc Bonferroni test.; RLU -relative luciferase unit.

Figure S8 .
Figure S8.Resveratrol and SAHA do not increase levels of Daughterless, TCF4 and Daughterless target genes Synapsin and discs large 1.A -rat cortical neurons were treated 50 µM resveratrol, 5 µM SAHA or 0.1% DMSO as control for 24h.Cells were lysed in 2x SDS sample buffer and equal amounts of protein extract was loaded to gel.For visualizing TCF4 ITF-2 Antibody (C-8) from Santa Cruz was used and the signals were normalized using β-tubulin signals, n=2.B -representative Western blot from rat cortical neurons.C -0-24 h old 3xFLAG-da flies were fed with 400 μM resveratrol or 2 μM SAHA, or 1% ethanol or 0.1% DMSO as control for 5 days.5 fly heads were lysed in 2x SDS sample