Maturation of a central brain flight circuit in Drosophila requires Fz2/Ca2+ signaling
- Tata Institute of Fundamental Research, India
Figures
Figure 1 with 2 supplements
dFz2 function is required in dopaminergic neurons during development for normal adult flight.
(A) Percentage flight times of individuals after knockdown of dFz2 in aminergic neurons (DdcGAL4), serotonergic neurons (TRHGAL4), dopaminergic neurons (THGAL4). Knockdown of dFz2 in aminergic …
Figure 1—figure supplement 1
Normal flight in flies with knockdown of dFz2 in non-dopaminergic neurons.
Percentage flight times of individual flies after knockdown of dFz2 in glutamatergic (OK371GAL4), mostly GABAergic (GADGAL4) and a peptidergic neuron subset (P386GAL4). Knockdown of dFz2 in these …
Figure 1—figure supplement 2
Expression of multiple dFz2-IR strains in dopaminergic neurons exhibits flight defects.
(A) Percentage flight times of dFz2-IR (BL27568, BL31390, BL31312) heterozygotes (gray bars) and flies with knockdown of dFz2 in dopaminergic neurons (red bars). Knockdown was achieved by …
Figure 2 with 3 supplements
RNAi-mediated knockdown of dFz2 function in Protocerebral Anterior Medial (PAM) dopaminergic neurons causes flight deficits.
(A) Expression pattern of THGAL4 in the anterior and posterior regions of the brain are shown. Dotted line markings show the neuronal clusters. PAM: protocerebral anterior medial; PAL: protocerebral …
Figure 2—figure supplement 1
Electrophysiological traces showed reduced firing upon knockdown of dFz2.
Representative traces for electrophysiological recordings obtained from DLMs of flies of the indicated genotypes.
Figure 2—figure supplement 2
Expression of dFz2 in PAM dopaminergic neurons.
Expression of GFP (Anti GFP; green) and dFz2 (Anti Fz2; red) in PAM dopaminergic neurons of R58E02GAL4> mCD8GFP flies and R58E02GAL4> mCD8GFP; dFz2-IR flies. dFz2 immunostaining was absent upon …
Figure 2—figure supplement 3
Expression of TH in PAM dopaminergic neurons during development.
Expression of GFP (Anti GFP; green) and TH (Anti TH; red) in the larval and pupal brain of R58E02GAL4>mCD8GFP organisms are shown. Separate confocal stacks for the anterior and posterior regions of …
Figure 3 with 3 supplements
dFz2 function is mediated through G-protein Go and IP3-mediated calcium signaling in dopaminergic neurons.
(A) A schematic showing dFz2-mediated activation of Go followed by IP3R-mediated Ca2+ signaling pathway and Store-operated Ca2+ entry (SOCE) through dSTIM and dOrai. Red (down-regulation) and green …
Figure 3—figure supplement 1
The canonical Fz2/β catenin signaling pathway does not function downstream of dFz2 in the context of flight circuit maturation.
(A) A schematic of canonical Wnt/dFz2 signaling. Upon activation by Wnt, in collaboration with a co-receptor (lipoprotein receptor-related protein or LRP5/6; encoded by gene Arrow in Drosophila), …
Figure 3—figure supplement 2
Go functions downstream of dFz2 in the context of flight circuit maturation.
Percentage flight times are shown for flies with dopaminergic specific expression of UAS-PTX.16 (red bars) at the indicated developmental stages obtained by temporal activation of THGAL4; GAL80ts. …
Figure 3—figure supplement 3
Non-canonical dFz2/Ca2+ signaling functions downstream of dFz2 in the context of flight circuit maturation.
(A) Schematic of putative non-canonical dFz2/Ca2+ signaling depicting the strategies used for testing this pathway. Red arrows represent knockdown and green arrows show over-expression of the …
Figure 4
Knockdown of dFz2 affects neuronal activity of maturing flight circuit PAM neurons.
(A) Percentage flight times of individual heterozygous controls (gray bars), dFz2 knockdown (dFz2-IR) in dopaminergic neurons (THGAL4) and PAM neurons (NP6510GAL4, R58E02GAL4) (red bars) followed by …
Figure 5 with 3 supplements
Expression of TH is reduced in PAM neurons by dFz2 knockdown.
(A) Expression of GFP (Anti GFP; green) and TH (Anti TH; red) is shown in PAM dopaminergic neurons marked by R58E02GAL4>mCD8GFP. (B) Significant reduction of GFP and TH immunoreactivity is observed …
Figure 5—figure supplement 1
Expression of GFP is altered upon expression of dFz2-IR in PAM neurons using R58E02GAL4.
(A) Scatter plot of the mean intensity of GFP expression in individual PAM neurons (N = 1280) from 16 brain hemispheres of the indicated genotypes (*p < 0.05, one-way ANOVA). (B) Kolmogorov-Smirnov …
Figure 5—figure supplement 2
Altered GFP expression was seen upon expression of dFz2-IR in dopaminergic neurons.
(A) mGFP expression in the PAM dopaminergic neurons observed in brains of animals of the indicated genotypes. The number of PAM dopaminergic neurons was significantly reduced upon expression of (B) d…
Figure 5—figure supplement 3
Knockdown of dFz2 in OK371GAL4-expressing neurons does not affect TH expression-positive PAM neurons.
Expression of GFP (Anti GFP; green) and TH (Anti TH; red) is shown in PAM neurons of the indicated genotypes. No change in the intensity of either GFP or TH immunostaining was observed upon …
Figure 6 with 1 supplement
Expression of DTH1 in PAM neurons rescues flight defects shown by dFz2 knockdown.
(A) Percentage flight times of individual heterozygous control flies (gray bars), flies with expression of dFz2-IR and THRNAi (dTH-IR) in PAM neurons (R58E02GAL4) (red bars), and flies with …
Figure 6—figure supplement 1
Expression of GFP is altered in PAM neurons upon knockdown of dFz2 in the presence of either DTH1 or dsDTH.
(A) Scatter plot of the mean intensity of GFP expression in individual PAM neurons (N = 1280) from 16 brain hemispheres of the indicated genotypes (*p < 0.05, one-way ANOVA). (B) Kolmogorov-Smirnov …
Figure 7 with 5 supplements
Mushroom body α’/β’ neurons regulate flight through Dop1R2.
(A) Percentage flight times of individual flies of the indicated genotypes. Flight defects were seen by reducing the activity of α’/β’ neurons (mb186bGAL4, red bar) and by knockdown of Dop1R2 in …
Figure 7—figure supplement 1
Dopamine receptor knockdown in MB neurons.
Percentage flight times of individuals upon knockdown of the indicated Dopamine receptors. The knockdowns were either in α’/β’ neurons (mb186bGAL4) or in α/β and γ neurons (mb247GAL4) of the …
Figure 7—figure supplement 2
Synaptic activity in α’/β’ lobes required for flight.
(A) Percentage flight times of individual flies of the indicated genotypes. Flight defects were seen by reducing the activity (30°C) of α’/β’ neurons using c305aGAL4 (*p < 0. 01, Mann–Whitney …
Figure 7—figure supplement 3
Knockdown of dFz2 does not affect climbing ability of flies.
Percentage climbers are shown for knockdown of dFz2 either in multiple dopaminergic neurons (THGAL4) or in the PAM dopaminergic cluster (R58E02GAL4). Blocking synaptic vesicle recycling by …
Figure 7—figure supplement 4
Maintenance of flight requires dFz2/Ca2+ signaling in dopaminergic neurons.
Long flight times monitored for 15 min are shown for the indicated genotypes.
Figure 7—figure supplement 5
Maintenance of flight requires Fz2/Ca2+ signaling in dopaminergic neurons.
Percentage of flies that do not initiate (0 s) or fly for time periods within the binned intervals (20 s each) is shown for the indicated genotypes.
Videos
Video 1
dFz2 knockdown in dopaminergic neurons result in flight defect.
Real time video recording of air-puff induced flight in the following genotypes from left to right. (1) THGAL4/+, (2) THGAL4;dFz2-IR, (3) dFz2-IR/+. Following a gentle air-puff THGAL4;dFz2-IR flies …
Video 2
dFz2 knockdown in PAM neurons result in flight defect.
Real time video recording of air-puff induced flight in the following genotypes from left to right. (1) R58E02GAL4/+, (2) R58E02GAL4;dFz2-IR, (3) dFz2-IR/+. Following a gentle air-puff R58E02GAL4;dFz…
Video 3
Overexpression of IP3R in dopaminergic neurons rescues flight defects of dFz2 downregulation.
Real time video recording of air-puff induced flight in the following genotypes from left to right. (1) THGAL4;dFz2-IR;itpr+, (2) THGAL4;dFz2-IR, (3) dFz2-IR/+. Following a gentle air-puff THGAL4; …
Video 4
Flight defects in dFz2 knockdown individuals can be rescued by over-expression of dSTIM.
Real time video recording of air-puff induced flight in the following genotypes from left to right. 1) THGAL4; dFz2-IR; dSTIM+, 2) THGAL4; dFz2-IR, 3) dFz2-IR/+. Following a gentle air-puff THGAL4; …
Video 5
Increased neuronal activity in PAM neurons rescues flight in individuals with dFz2 knockdown.
Real time video recording of air-puff induced flight in the following genotypes from left to right. (1) R58E02GAL4;dFz2-IR;NaChBac, (2) R58E02GAL4;dFz2-IR, (3) dFz2-IR/+. Following a gentle air-puff …
Video 6
DTH over-expression in PAM neurons rescues flight in individuals with dFz2 knockdown.
Real time video recording of air-puff induced flight in the following genotypes from left to right. (1) R58E02GAL4;dFz2-IR;DTH1, (2) R58E02GAL4;dFz2-IR, (3) dFz2-IR/+. Following a gentle air-puff R58…
Tables
Table 1
Summary of expression pattern of GAL4s
| THC’ | THC1 | THF2 | NP6510 | R58E02 | |
|---|---|---|---|---|---|
| PAM | + | ++ | − | ++ | +++ |
| PAL | + | + | − | − | − |
| PPM1 | + | ++ | − | − | − |
| PPM2 | + | + | + | − | − |
| PPM3 | − | − | + | − | − |
| PPL1 | − | + | ++ | − | − |
| PPL2 | + | + | + | − | − |
| T1 | + | + | − | − | − |
| T2 | − | + | + | − | − |
| T3 | − | + | − | − | − |
| Ab | + | + | + | − | − |
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Table summarizing the expression pattern of THC’GAL4, THC1GAL4, THF2GAL4, NP6510GAL4, and R58E02GAL4 in specified dopaminergic neuronal clusters. Clusters shown in Figure 2A and thoracic ganglion (T1, T2, T3, Ab) were examined for the expression. Plus (+) and minus (−) indicate the presence and absence of dopaminergic positive neurons, respectively. Double plus (++) and triple plus (+++) indicate the presence of >5 and >50 dopaminergic positive neurons, respectively. 10 brain hemispheres were analyzed for the expression.
