Modulation of light-driven arousal by LIM-homeodomain transcription factor Apterous in large PDF-positive lateral neurons of the Drosophila brain

To examine whether Ap is expressed in PDF neurons, we used ap::GFP knock-in flies, which express a GFP reporter in a pattern consistent with endogenous Ap expression29. We observed the colocalization of Ap::GFP and the nucleus-targeted mCherry reporter for PDF neurons in Pdf-GAL4/ap::GFP; UAS-mCherry.NLS/+ flies, and confirmed that Ap localizes to the nuclei of PDF neurons including l-LNvs and s-LNvs (Fig. 1). Among l-LNvs and s-LNvs, 3–5 cells with mCherry.NLS signals were detected in each brain hemisphere [l-LNvs, 4 ± 0.2 (mean ± SEM) cells, N = 8; s-LNvs, 3.6 ± 0.2 cells, N = 8], and among them, 2–4 cells were GFP-positive [l-LNvs, 3 ± 0.4 cells, N = 8; s-LNvs, 2.4 ± 0.4 cells, N = 8].

First, we examined whether ap expression is required in neurons for the expression of the proper sleep/wake phenotype. We knocked down ap in neurons by expressing ap RNAi using the pan-neuronal driver nSyb-GAL4 and analyzed sleep/wake behaviors under LD conditions. Pan-neuronal ap knockdown reduced the amount of sleep during both the day and night (Fig. 2a,b, green circles and bars). However, parameters for daytime and nighttime sleep are differentially affected by ap knockdown. For example, compared with control flies (Fig. 2c, black and gray bars), pan-neuronal knockdown of ap expression decreased sleep-bout duration but did not have a significant effect on wake-bout duration in the night (Fig. 2c,d, green bars). In contrast, ap knockdown increased wake-bout duration but kept sleep-bout duration unchanged during the day (Fig. 2c,d, green bars). These results indicate that pan-neuronal ap knockdown adversely affects sleep initiation during the day, while it disrupts sleep maintenance during the night. The waking activity index was slightly increased by ap knockdown during both the day and night (Fig. 2e, green bars). As seen in Fig. 2a, the sleep-suppressing effect of pan-neuronal ap knockdown was most significant during the daytime near dawn and dusk. Indeed, when the total waking time in the morning [Zeitgeber time (ZT) 0–4], midday (ZT4–8), and evening (ZT8–12) was compared between ap-knockdown flies and controls, differences were most obvious in the morning and evening (Fig. 2f).

To examine whether neuron-specific knockdown of ap affects circadian rhythms, we measured the locomotor activity of the nSyb-GAL4/UAS-ap RNAi flies for 10 days in DD and calculated the percentage of the flies showing rhythmic locomotor activity. Similarly to the control flies, nSyb-GAL4/UAS-ap RNAi flies showed rhythmic locomotor activity in DD (Fig. S2), indicating that ap knockdown does not affect circadian rhythms of locomotor activity.

Next, we examined the significance of PDF neurons in the wake-promoting effect of pan-neuronal ap knockdown by specifically abolishing GAL4 activity in PDF neurons using Pdf-GAL8024. The effect of pan-neuronal knockdown of ap was considerably mitigated when Pdf-GAL80 was included (Fig. 2a–f, orange circles and bars). This result suggests that Ap expression in PDF neurons is required for proper sleep/wake regulation, while Ap-positive non-PDF neurons also play a role. In all subsequent experiments, we focus on the total sleep amount (day and night) and waking time (morning, midday, and evening), because these sleep/wake parameters are most significantly affected by ap knockdown in a PDF neuron-dependent manner.

The significance of Ap expression in PDF neurons was directly accessed by ap knockdown in a PDF neuron-specific manner using Pdf-GAL4 in combination with UAS-ap RNAi. Under LD conditions, the targeted expression of ap RNAi to PDF neurons also induced the characteristic sleep/wake phenotype that was observed for pan-neuronal ap knockdown. The amount of sleep during the day and night was reduced (Fig. 3a,b), and the total waking time in the morning, midday and evening increased (Fig. 3c). To confirm that the observed effects depend on ap knockdown, we examined Pdf-GAL4/UAS-dOrk1Δ NC flies as negative controls. dOrk1ΔNC is a nonfunctional isoform containing a K⁺ channel-inhibiting mutation; and it is known that induction of this isoform in specific neurons does not affect neural activity30. As we expected, dOrk1ΔNC expression in PDF neurons affected neither the sleep amount nor waking time (Fig. S3).

Genetic ablation of l-LNvs increases the amount of sleep in LD cycles, but this phenotype disappears after the transfer of flies from LD to DD conditions28. In addition, excitation of l-LNvs promotes arousal26,28. Thus, PDF-producing l-LNvs play a key role in light-driven arousal. To test whether the high-arousal phenotype in ap-knockdown flies is also light-dependent, we examined Pdf-GAL4/UAS-ap RNAi flies for their sleep/wake phenotype in DD. Compared with control flies, no significant differences were detected in sleep amount during the day and night or waking time in the morning [Circadian time (CT) 0–4], midday (CT4–8) and evening (CT8–12) (Fig. 3d–f), indicating that the wake-promoting effect induced by ap knockdown in PDF neurons is light-dependent. These results revealed that endogenous Ap in PDF neurons buffers light-driven arousal in wild-type flies.

To further examine how disruption of the Ap function affects sleep/wake behaviors, we used two truncated forms of Ap (Ap^ΔLIM and Ap^ΔHD; Fig. 4a). They are expected to act as a dominant negative protein and inhibit the transcriptional activity of the Ap/Chi complex because (1) Ap^ΔHD lacking a HD can reduce the amount of the functional Ap/Chi complex by sequestering endogenous Chi (Fig. 4b), and (2) Ap^ΔLIM lacking two LIM domains can interfere with the DNA binding of the Ap/Chi complex (Fig. 4c). In fact, Ap^ΔHD was shown to induce the dominant negative effect during wing development4. Here, we generated two transgenic lines, UAS-ap^ΔHD and UAS-ap^ΔLIM, and examined the effects of Ap^ΔHD and Ap^ΔLIM on sleep/wake behaviors. PDF neuron-specific expression of Ap^ΔHD induced a significant decrease in sleep amount only during the day (Fig. 4d,e). As was observed in Pdf-GAL4/UAS-ap RNAi flies, morning and evening waking times increased (Fig. 4f). Similar dominant-negative effects on the sleep/wake phenotype were detected when Ap^ΔLIM was expressed in PDF neurons (Fig. 4g–i). These results further confirmed our finding that disruption of the Ap function promotes arousal during the day.

Considering the dominant negative effects of Ap^ΔHD and Ap^ΔLIM in the sleep/wake phenotype, it is possible that the transcriptional activity of Ap/Chi is crucial to arousal regulation during the day. To investigate this possibility, we first examined whether the knockdown of Chi in PDF neurons also affects sleep/wake behaviors. During the day, Pdf-GAL4/UAS-Chi RNAi flies showed reduced sleep amount and lengthened waking time in the morning and evening (Fig. 4j–l). Next we used two dominant-negative forms of Chi (Chi^ΔLID and Chi^ΔDD; Fig. S4). The transcriptional activity of Ap/Chi is expected to be inhibited by the induction of these truncated forms because they can interfere with the formation of the functional Ap/Chi tetramer. Actually, expression of Chi^ΔLID or Chi^ΔDD induces the dominant negative effects in wing development or posteclosion behavior requiring Chi functions12,31. As was observed in Pdf-GAL4/UAS-Chi RNAi flies, the targeted expression of Chi^ΔLID in PDF neurons reduced sleep amount during the day and increased waking time in the morning and evening (Fig. S5a–c). The expression of Chi^ΔDD induced weak but significant reduction in the amount of daytime sleep and lengthened the morning waking time (Fig. S5d–f). Thus, disruption of the Chi function, as well as Ap dysfunction, also promotes arousal during the day.

Ap is expressed in PDF-positive s-LNvs and l-LNvs (Fig. 1). Considering the effects of the PDF neuron-specific knockdown on daytime sleep and circadian rhythm, it is likely that Ap plays an important role in light-activated wake-promoting neurons, l-LNvs. To examine this possibility, we used a GAL4 line, c929, which expresses GAL4 in peptidergic neurons including l-LNvs25,32. As previously reported, GFP signals were detected in l-LNvs, but not in s-LNvs, in c929/UAS-mCD8::GFP flies (Fig. 5a,b). As was observed in Pdf-GAL4/UAS-ap RNAi flies, c929/UAS-ap RNAi flies also showed reduced sleep amount and increased waking time in the morning and evening (Fig. 5c–f, green circles and bars).

To examine the significance of Ap expression in l-LNvs in the regulation of daytime arousal, we used c929 in combination with Pdf-GAL80. We confirmed that the GFP signals in l-LNvs were abolished in c929 with Pdf-GAL80 (Fig. 5g,h). When c929 GAL4 activity was suppressed in PDF neurons expressing Pdf-GAL80, the total sleep and waking times in the morning and midday were not significantly altered in comparison with c929/UAS-ap RNAi flies (Fig. 5c–e, orange circles and bars), but the evening waking time significantly decreased (Fig. 5e). In addition, regarding the waking time between ZT0 and ZT2, no significant differences were detected between c929/UAS-ap RNAi Pdf-Gal80 and c929/+ control flies (Fig. 5f, orange bar). Thus, our results indicate that the enhanced-arousal phenotype in c929/UAS-ap RNAi flies is nearly rescued in c929/UAS-ap RNAi Pdf-GAL80 flies. We examined whether Ap::GFP is expressed in c929-positive non-PDF neurons using the c929/ap::GFP; UAS-IVS-mCD8::RFP/+ flies. We confirmed that Ap is expressed in several c929-positive cells located in the pars intercerebralis (PI), subesophageal zone (SEZ), and some neurons in the posterior brain region (Fig. S6), suggesting that these neurons may partially contribute to the enhanced-arousal phenotype in c929/UAS-ap RNAi flies.

We next used a GAL4 line, Mai179, which predominantly expresses GAL4 in s-LNvs and weakly expresses GAL4 in one or two cells of l-LNvs33. ap knockdown in Mai179-positive neurons did not affect sleep amount or waking time (Fig. S7, green circles and bars). Taken together, our results indicate that l-LNvs are predominantly responsible for the wake-promoting effect induced by ap knockdown.

Genetic ablation of l-LNvs inhibits light-driven arousal28 and excitation of l-LNvs promotes arousal26,28. In addition, Pdf null mutant flies show increased amount of daytime sleep26. These observations indicate that PDF production and release from l-LNvs promote light-driven arousal. We next examined whether Ap downregulates Pdf expression because some LIM-HD proteins act as a transcriptional repressor1,34. In qRT-PCR analyses, no significant difference was detected in Pdf mRNA expression levels between Pdf-GAL4/UAS-ap RNAi and control flies (Pdf-GAL4/+) (Fig. 6a). In addition, no significant difference was detected in PDF immunoreactivity in the cell bodies of l-LNvs between c929/UAS-ap RNAi and control flies (c929/ UAS-GFP RNAi) (Fig. 6b). Taken together, it is unlikely that PDF neuron-specific ap knockdown inhibits PDF expression. In addition, compared with control flies (Pdf-GAL4/UAS-GFP RNAi), no prominent structural defects in PDF-positive neurons was detected in Pdf-GAL4/UAS-ap RNAi flies (Fig. 6c,d), and ap knockdown did not affect the number of l-LNvs (Fig. 6e).

In Drosophila, l-LNvs project to the optic lobe and many PDF-positive varicosities, which are considered the sites of PDF release, exist in the optic lobe35. Thus, it is also possible that ap knockdown promotes arousal as a result of overproduction of PDF-releasing sites of l-LNvs. To address this possibility, we compared the number of PDF-positive varicosities in the optic lobe between Pdf-GAL4/UAS-ap RNAi and Pdf-GAL4/UAS-GFP RNAi flies. The mean number of PDF-positive varicosities of 0.5, 1, or 2 μm diameter was counted in each genotype. No significant difference was detected between Pdf-GAL4/UAS-ap RNAi and control flies (Fig. 6f–h), indicating that ap knockdown does not induce overproduction of PDF releasing sites of l-LNvs.

To determine whether transient ap knockdown in l-LNvs promotes arousal during the adult stage, we employed the TARGET system. Using UAS-ap RNAi/c929; tub-GAL80^ts/+ flies, we performed temperature shift experiments (22 °C–30 °C–22 °C) as shown in Fig. S8a. First, we calculated daytime and nighttime sleep. In all genotypes, increase in the amount of daytime sleep and decrease in that of nighttime sleep were apparent at the restrictive temperature (Fig. S8b, L5; S8c, D4 and D5), indicating that the temperature shift itself modifies sleep/wake behaviors regardless of the genotype. Here, we calculated waking index (see Supplementary Material and Methods) to estimate the efficacy of transient ap knockdown for sleep/wake behaviors. In the morning (ZT0–4), midday (ZT4–8), and evening (ZT8–12), the waking index was defined as the difference between the mean waking time of GAL4 control flies (c929/+) and the waking time of each individual in UAS-ap RNAi/c929; tub-GAL80^ts/+ or UAS control (UAS-ap RNAi/+; tub-GAL80^ts/+) flies. Finally, mean waking index was calculated. In UAS-ap RANi/c929; tub-GAL80^ts/+ flies, the mean waking index in the morning at the restrictive temperature was significantly higher than that at the permissive temperature (Fig. S8d, green bars, L5), but not in control flies (Fig. S8d, gray bars). Unlike the mean waking index in the morning, those in the midday and evening did not increase after a temperature shift (Fig. S8e and f, green bars, L5). Thus, these results suggest that at least morning arousal is promoted by transient ap knockdown. In UAS-ap RANi/c929; tub-GAL80^ts/+ and UAS control flies, the mean waking index in the evening at the restrictive temperature significantly decreased in comparison with that during the first permissive temperature exposure (Fig. S8f, gray and green bars, L4 and L5). Thus, this reduction is not due to transient ap knockdown. However, in UAS-ap RANi/c929; tub-GAL80^ts/+ flies, the mean waking index in the evening during the second permissive temperature exposure (Fig. S8f, grenn bars, L6–L8) recovered to the mean waking index during the first permissive temperature exposure (Fig. S8f, grenn bars, L4), but not in UAS control flies (Fig. S8f, gray bars). Although the reason for this differential recovery remains unclarified, it may be due to the combined effects of the gradual recovery of ap expression in c929-positive cells, the impact of the temperature shift itself (i.e., the shift modifies the physiological state of flies), and the genetic background of UAS-ap RANi/c929; tub-GAL80^ts/+ flies.

In the genome-wide expression analysis using all transcripts from the fly head, Claridge-Chang et al.36 have revealed that ap expression shows 24 h oscillation under LD conditions and the expression level of ap mRNA peaks detected in the middle of the night (around ZT16)36. Here, we examined whether Ap expression in l-LNvs also shows daily rhythms in LD cycles using c929/ap::GFP; UAS-mCherry.NLS/+ flies. The fluorescence intensity of Ap::GFP and mCherry.NLS in l-LNvs was measured at ZT0, ZT6, ZT12, and ZT18. Although the absolute mCherry.NLS fluorescence intensity level in l-LNvs did not change within 1 day (Fig. 7a,b), absolute and relative Ap::GFP levels increased during the night and peaked at ZT18 (Fig. 7c,d). To examine whether this oscillation is light-dependent, we next measured Ap::GFP levels in DD. As shown in Fig. 7e–h, clear oscillation of Ap::GFP expression was not detected in l-LNvs in DD (Fig. 7e–h). Taken together, we concluded that daily rhythms of Ap expression in l-LNvs is generated by a direct light-dependent mechanism not the circadian clock.

Fly stocks used for this study are as follows: wild-type Drosophila melanogaster Canton-S (CS), ap::GFP [Bloomington stock center (BS), #38423], Pdf-GAL4 (BS, #6900), nSyb-GAL4 (BS, #51941), c929 (BS, #25373), Mai179 (obtained from Dr. Orie T. Shafer, University of Michigan), Pdf-GAL80 (obtained from Leslie C. Griffith, Brandeis University), UAS-ap RNAi (NIG-fly, 8376R-1), UAS-ap^ΔHD (see next section), UAS-ap^ΔLIM (see next section), UAS-Chi RNAi (VDRC, 43934), UAS-Chi^ΔLID (obtained from Dr. Veronica Rodriguez, Tata Institute of Fundamental Research), UAS-Chi^ΔDD (obtained from Dr. Veronica Rodriguez), UAS-dORK1ΔNC (BS, #6587), UAS-mCherry.NLS (BS, #38424), UAS-IVS-mCD8::RFP (BS, #32218), and UAS-GFP RNAi (NIG-fly, GFP-IR-2). Flies were raised on glucose-yeast-cornmeal medium at 25.0 ± 0.5 °C in a 12-h light:12-h dark (LD) cycle. All lines except for UAS-mCherry.NLS and UAS-IVS-mCD8::RFP were outcrossed for at least six generations to white flies with the CS genetic background.

Full-length ap cDNA was isolated by RT-PCR using adult fly head RNA and two primers, 5′-GCGGCCGCCAAAATGGGCGTCTGCACCGAGGAGCGC-3′ and 5′TCTAGATTAGTCCAAGTTAAGTGGCGGTGTGC-3′.

The PCR product was digested with NotI and XbaI, and cloned into a pBluescript (pBS) II SK(+). Constructs of ap^ΔHD and ap^ΔLIM were generated by self-ligation of the PCR products amplified from the ap cDNA-containing pBS II SK(+) using two primer sets as follows: ap^ΔHD –forward, 5′-ATGATGAAGCAGGATGGCAGCGGC-3′; ap^ΔHD –reverse, 5′-CGACGAGGAGCTTAGGTGCGAGCC-3′; ap^ΔLIM –forward, 5′-GGGGATACCGCCTCATCCAGTATG-3′; ap^ΔLIM –reverse, 5′-GAGGTTGCGCGTTATTTTGCTATC-3′. The fragments lacking nucleotides 436–807 (ap^ΔLIM) and 1096–1275 (ap^ΔHD) of ap cDNA were obtained by restriction enzyme digestion using NotI and XbaI, and then they subcloned into the NotI/XbaI-digested pUAST attB vector54. Constructs of UAS-ap^ΔHD and UAS-ap^ΔLIM were injected into the eggs of PBac{y[+]-attP-9A}VK00005 (BS, #24862).

Two- to three-day-old adult male flies were individually placed in a glass tube (5 mm × 65 mm) with fly food and monitored in a 12-h L:12-h D (LD) cycle (lights on at 8:00) at 25 °C. The locomotor activity of individual flies was analyzed using the DAM system (Trikinetics). Flies were acclimated in the glass tubes for 3 days in LD cycles before measurement of sleep. Locomotor activity data were collected at 1-min intervals for 3 days and analyzed with a Microsoft Excel-based program as described previously55. Sleep was defined as 5 min or more of behavioral inactivity, as previously described56. Total sleep amount, sleep- and wake- bout durations, waking activity, and waking time in the morning (ZT0–4 or CT0–4), midday (ZT4–8 or CT4–8) and evening (ZT8–12 or CT8–12) were analyzed for each 12-h period of LD or DD and averaged over 3 days for each condition. The waking activity was calculated by dividing the total activity counts during the length of the wake period during the day and night as reported previously57.

The Kolmogorov–Smirnov test was used to estimate whether the data are normally distributed. When the data were not distributed normally, we carried out the log transformation of the data. When the basic data or transformed data are distributed normally, one-way ANOVA followed by post-hoc analysis using Scheffe’s test was carried out for multiple pairwise comparisons. For multiple group analysis of the nonparametric data, we used nonparametric ANOVA (Kruskal–Wallis test) followed by the rank-sum test for multiple pairwise comparisons. The computer software IBM SPSS statistics22 (IBM Japan, Ltd.) was used for these tests.

The flies were entrained to 12:12 LD cycles during their development, and 1-d-old single males were placed in glass tubes containing standard food, and their activity was monitored using the DAM system (Trikinetics). Infrared beam crosses in 30 min bins were recorded. We examined the effects of panneural knockdown of ap on circadian locomotor rhythms. Activity was monitored for 4–5 days of LD at 25 °C, followed by 10 days of DD at 25 °C. The circadian period and rhythmicity were estimated from the data of locomotor activity collected for 10 days of DD. Significant circadian rhythmicity was defined as the presence of a peak in periodogram power that extends above the significance line (P < 0.05) in chi-square analysis. Clocklab software (Actimetrics) was used to analyze the circadian period and rhythmicity.

Using TRizol (Invitrogen), total RNA was isolated from approximately 30 male fly heads of each genotype. cDNA was synthesized by a reverse transcription reaction using a QuantiTect Reverse Transcription Kit (QIAGEN). Real-time quantitative PCR was carried out using THUNDERBIRD SYBR qPCR Mix (TOYOBO) and a Chromo 4 Detector (MJ Research, Hercules, CA). Expression levels of Pdf mRNA were normalized by those of rp49 mRNA. The average normalized Pdf mRNA expression levels in control flies was calculated using data from five independent assays. The ratio of normalized Pdf mRNA expression level in each experimental genotype to the average control value was calculated. The mean (± SEM) ratio was calculated for data from five independent assays. The primer sequences used for real-time PCR were as follows: Pdf –forward, 5′-ATCGGGATCTCCTCGACTGG-3′; Pdf –reverse, 5′-ATGGGCCCAAGGAGTTCTCG-3′; rp49–forward, 5′-AAGATCGTGAAGAAGCGCAC-3′; rp49–reverse, 5′-TGTGCACCAGGAACTTCTTG-3′.

Adult male brains (over 5 days old) were fixed in PBS containing 4% formaldehyde for 45–60 min at room temperature. After three washes in PBST (0.1–0.2% Trion X-100 in PBS), they were blocked for 1 h in 1% normal goat serum in PBST and then incubated with a primary antibody. Next, they were incubated with a secondary antibody for 24 h at 4 °C after three washes in PBST.

For PDF staining, brains were stained with a mouse anti-PDF antibody (The Developmental Studies Hybridoma Bank at the University of Iowa, 1:2000), and Alexa Fluor 568 anti-mouse IgG (Invitrogen A11004) was used as the secondary antibody (1:1000). For GFP staining, brains were stained with a rabbit anti-GFP antibody (Invitrogen A11122, 1:200), and Alexa Fluor 488 anti-rabbit IgG (Invitrogen, A11008) was used as the secondary antibody (1:1000). Fluorescence was observed under a confocal microscope (Carl Zeiss LSM710 and Nikon C2).

To examine whether ap knockdown in l-LNvs affects PDF-positive cell number or PDF immunoreactivity, c929/UAS-ap RNAi flies (6 days old) were used. c929/UAS-GFP RNAi flies were used as the control. The brain was dissected between ZT0 and ZT2. A confocal image stack of the brain hemisphere containing l-LNvs and s-LNvs was Z-projected into several sequential sections. Z-sections were collected at 0.53 μm intervals. We counted the number of PDF-positive cell bodies, and then the PDF immunoreactivity was quantified in a manually set region of interest (ROI) of the cell body region in each l-LNv and s-LNv using a histogram tool of ZEN 2010 software (Carl Zeiss). To compensate for the differences in fluorescence intensity between different ROIs, PDF immunoreactivity in l-LNvs was normalized to that in s-LNvs because c929 does not induce GAL4-dependent gene expression in s-LNvs. In all samples, the image data were acquired under identical conditions. The mean relative PDF immunoreactivity was calculated for each genotype. Student’s t-test was used for pair-wise comparison.

Quantitative analysis of PDF-positive varicosities was conducted in the Pdf-GAL4/UAS-ap RNAi and control (Pdf-GAL4/UAS-GFP RNAi) flies. The brain was dissected between ZT0 and ZT2. A confocal image stack of one side of optic lobes was Z-projected (1.0 μm intervals). The PDF-positive varicosities on one side of the optic lobes of each brain were counted using the spot detection algorithm in Imaris software 7.1.0 (Bitplane). The diameters of spots were set at 0.5, 1, and 2 μm, and the number of spots was counted for each diameter. The mean number of varicosities was calculated for each genotype. Student’s t-test was used for pair-wise comparison.

c929/ap::GFP; UAS-mCherry.NLS/+ flies (6-d-old) were used to measure Ap::GFP expression level in l-LNvs. For the measurement of Ap::GFP level in LD cycles, adult male brains were collected at 4 time points (ZT0, 6, 12, and 18) after the flies were entrained to 6 LD cycles. For the measurement of Ap::GFP level in DD, adult male brains were collected at 4 time points (CT0, 6, 12, and 18) on the third day of DD after the flies were entrained to 3 LD cycles. After extraction of the brains, they were stained with a rabbit anti-GFP antibody as described above. A confocal image stack of the brain hemisphere containing mCherry. NLS-positive- and Ap::GFP-positive-l-LNvs was Z-projected into 5–7 sequential sections (0.53 μm intervals). mCherry.NLS and Ap::GFP levels in l-LNvs were measured on the basis of mCherry or GFP fluorescence intensity determined using the colocalization tool of ZEN 2010 software (Carl Zeiss). Fluorescence intensity was measured in a manually set ROI of the mCherry.NLS-positive nuclear region in each l-LNv. To compensate for differences in fluorescence intensity between different ROI, Ap::GFP fluorescence intensity was normalized to the fluorescence intensity of mCherry.NLS. For all samples, their image data were acquired under identical conditions. Using the computer software BellCurve for Excel (Social Survey Research Information Co., Ltd.), nonparametric ANOVA (Kruskal–Wallis test) followed by post-hoc analysis using the Steel-Dwass test was carried out for multiple pairwise comparisons.