The roles of anillin in the Drosophila nervous system

doi:10.21203/rs.3.rs-3968358/v1

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The roles of anillin in the Drosophila nervous system

https://doi.org/10.21203/rs.3.rs-3968358/v1

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Anillin (Ani) is an evolutionarily conserved protein with a multi-domain structure that cross-links cytoskeletal proteins and plays an essential role in the formation of the contractile ring during cytokinesis. However, Ani is highly expressed in the human central nervous system (CNS), which does not actively divide. Moreover, it scaffolds myelin in the CNS of mice and modulates neuronal migration and growth in Caenorhabditis elegans. This protein is also highly expressed in the Drosophila CNS. However, its role remains unclear. In the present study, Ani was highly expressed in type I and II neuroblasts, whereas it was poorly expressed in the neuromuscular junction (NMJ), axons, and some neurons in the ventral nerve cord. In addition, neuron-specific ani knockdown flies had a short lifespan and larval locomotor defects, along with an abnormal morphology of the NMJ, learning disability, and a swollen CNS. These results show that Ani plays important roles not only in proliferating cells, but also in the Drosophila nervous system.

Biological sciences/Developmental biology/Cell proliferation

Biological sciences/Neuroscience/Neurogenesis

Anillin (Ani) was originally isolated from Drosophila embryos as an F-actin-binding protein [1]. It is a scaffold protein conserved from yeast to humans, with a multi-domain structure that comprises myosin-binding, F-actin-binding, anillin homology, and pleckstrin homology (PH) domains. This protein forms the contractile ring by recruiting septins via the PH domain during cytokinesis [2–4]. Additionally, its subcellular localization changes drastically during the cell cycle in proliferating cells. Although Ani is localized to the cytoplasm and cortex during metaphase and subsequently recruited to the prospective contractile ring during anaphase, it is mainly found in the nucleus during interphase [5]. This protein is expressed in tissues with actively dividing cells, such as the lungs, bone marrow, and testes. Furthermore, its function during cytokinesis has been extensively studied. Ani has crucial functions in the regulation of cell-cell junction integrity [6–9] and is highly expressed in the human brain [10], even in non-dividing cells and tissues. Ani stabilizes F-actin at the leading edge of neurons during neuronal migration [11] and facilitates septin assembly to prevent abnormal myelin outfolding [12, 13]. However, its precise functions in the nervous system remain unclear.

Similar to humans, the larval central nervous system (CNS) is among the most ani mRNA-expressing tissues[14]. Although Ani contributes to the asymmetric division of the peripheral nervous system [15], its role in the Drosophila CNS has not been investigated. Therefore, this study comprehensively investigated the expression pattern of Ani in the larval nervous system and its role in the nervous system using pan-neuron-specific ani knockdown. Ani is highly expressed in dividing cells, including neuroblasts (NBs) in the brain lobe. However, it was poorly expressed in some neurons in the ventral nerve cord, axons, and neuromuscular junctions (NMJ) of motor neurons. In addition, it was hypothesized that Ani is localized along the F-actin in the axon. Furthermore, the knockdown of ani in the nervous system significantly perturbed larval learning, larval and adult locomotor ability, and viability. These results indicate that Ani plays an important role in the Drosophila nervous system.

Expression of ani in the larval nervous system

The ani gene is predicted to be relatively highly expressed in the larval CNS based on the RNA-seq analysis and to have three splice variants (SV-A, SV-B, and SV-C) in the Drosophila database, FlyBase (Fig. 1A) [16]. Therefore, we purified total RNA and proteins from the larval CNS and performed quantitative RT-PCR (qRT-PCR) and Western blot analyses to determine which splice variant was expressed in the larval CNS. The qRT-PCR with the primer set designed to detect SV-C or both SV-B and C showed only marginal amplification of the PCR signal. In contrast, qRT-PCR with the primer set designed to detect both SV-A and B showed extensive amplification of the signal (Fig. 1A, B), which is similar to results reported in early embryos [17]. These results suggest that SV-A is predominantly expressed in the larval CNS. In Western blot analysis with anti-Ani IgG, the 178 kDa band corresponding to Ani predicted to be encoded by SV-A was predominantly detected (Fig. 1C). The marginally detected 87 kDa and approximately 75 kDa bands may have been derived from SV-C or the degradation products of the 178 kDa product. These results were consistent with those of the qRT-PCR, indicating that SV-A is predominantly expressed in the larval CNS. Next, we performed immunofluorescence staining of the larval nervous system with anti-Ani IgG to comprehensively investigate Ani expression patterns. Ani signals were predominantly detected in proliferating cells of the CNS (Figs. 1, S1). In particular, this protein was highly expressed in the NBs marked by Deadpan (Dpn) in the brain lobe, ventral nerve cord (VNC), neural epithelium, and some lamina cells in the brain lobe (Fig. 1D). In contrast, Ani showed a mutually exclusive expression pattern with Dachshund (Dac), except for a few layers of cells next to the lamina furrow (LF) (Fig. 1D e, f). Although Dac is a marker for lamina precursor cells and neurons [18, 19], Dac and Ani were co-expressed in the proliferating cells that form a cell layer next to the LF in the outer proliferation center (Fig. 1D f).

Although most of the Ani was expressed in the cell nuclei of the CNS, except in cells expressing embryonic lethal abnormal vision (Elav), a neuronal marker (Figs. 2A, S2), Ani was also detected weakly in the nuclei of Elav-expressing cells in the optic lobe and VNC (Fig. 2B, S2). In addition, it was detected at the synaptic boutons in the neuromuscular junction (NMJ) (Fig. 2C) and axons (Figs. 2D, S3), whereas the strong and large signals in the PNS corresponded to the glial nuclei (Fig. 2D). Furthermore, the Ani dot signals in the axon were connected by a filament-like structure and were localized between the plasma membrane and neural lamella, where perineurial and subperineurial glia are located outside the neurons (Figs. 2D, S3) [20]. These results suggest that Ani functions, not only in proliferating cells, but also in non-proliferating cells in the Drosophila larval nervous system.

ani knockdown in the CNS induces defective neurological phenotypes

We knocked down ani using the elav-GAL4 driver, a pan-neuronal driver, or OK371-GAL4, a glutamatergic neuronal GAL4 driver [21], combined with two independent ani-RNAi lines to investigate the role of ani in the neurons of the Drosophila nervous system. We then analyzed the phenotypes of the knockdown flies (Figs. 3, S3). The expression levels of ani mRNA and Ani in the CNS were evaluated using qRT-PCR, Western blotting, and immunostaining with anti-Ani IgG. ani mRNA levels decreased to 30% and 19% in elav > ani-IR³³⁴⁶⁵ and elav > ani-IR⁵³³⁵⁸, respectively (Fig. 3A). Similarly, Ani levels in the CNS of the ani knockdown larvae decreased (Figs. 3B, C, S4). Furthermore, the target sequences of ani-IR³³⁴⁶⁵ and ani-IR⁵³³⁵⁸ corresponded to nucleotide positions 616–953 and 597–617 of SV-A, respectively, which overlap by only two nucleotides. Data from a BLAST search against the EST database and information on the Vienna Drosophila Resource Center (VDRC) homepage predicted no off-target effect in these IR strains. These results indicate that the knockdown efficiency was higher for elav > ani-IR⁵³³⁵⁸ than for elav > ani-IR³³⁴⁶⁵. We investigated the larval learning and locomotor ability of the pan-neuronal ani knockdown flies to elucidate the role of ani in the nervous system. First, we performed an odor-taste associative learning assay with early-stage third-instar larvae to examine their learning ability [22]. Although Drosophila larvae showed no preference between amylacetate (AM) and octanol (OCT) without training, control larvae learned the association between odors and rewards. Thus, we calculated a learning index based on the AM preference after training. The learning index considerably decreased in elav > ani-IR⁵³³⁵⁸ larvae and slightly decreased in elav > ani-IR³³⁴⁸⁵ larvae. However, the difference was not statistically significant (Fig. 3D). Next, we performed a crawling assay to investigate the effect of ani knockdown on larval locomotor abilities. elav > ani-IR⁵³³⁵⁸ and elav > ani-IR³³⁴⁸⁵ larvae showed significantly reduced locomotor ability and slightly decreased crawling speed, respectively (Figs. 3E, F).

Although most elav > ani-IR³³⁴⁶⁵ flies survived until adulthood, almost all elav > ani-IR⁵³³⁵⁸ died during the pupal stage. All escapers, albeit few, were considerably weak and died within a day after eclosion. The median longevity of ani knockdown flies was 28 d (elav > ani-IR³³⁴⁶⁵) and 1 d (elav > ani-IR⁵³³⁵⁸), which was markedly shorter than that of the control flies (38 d) (Fig. 3G). Next, we examined the locomotor ability of only elav > ani-IR³³⁴⁶⁵ adult flies using a climbing assay because the escapers in the elav > ani-IR⁵³³⁵⁸ group failed to climb the wall and died the day after eclosion. The climbing ability of three-day-old elav > ani-IR³³⁴⁶⁵ flies showed substantial defects (Fig. 3H). These results indicate that ani plays essential roles in elav-expressing cells of the Drosophila nervous system and that defective ani knockdown in elav > ani-IR⁵³³⁵⁸ flies is more severe than that in elav > ani-IR³³⁴⁶⁵ flies.

ani knockdown in the CNS leads to abnormal neuropil formation and increases the cell size

Because the mushroom body of the larval brain is the center of olfactory associative learning and memory ability [23–25], we conducted immunostaining of the CNS with anti-Brp IgG and anti-CadN IgG to visualize the mature neuropil [26, 27]. Both Brp and CadN expression patterns were abnormal in elav > ani-IR⁵³³⁵⁸ larvae, especially the size of the BRP area in the brain lobe and the distribution patterns of CadN in the optic lobe and thoracic ganglion in the VNC (Fig. 4A). The ratio of the neuropil area to the brain lobe area markedly decreased in both elav > ani-IR³³⁴⁶⁵ and elav > ani-IR⁵³³⁵⁸ larvae (Figs. 4B, C).

Elav was believed to be exclusively expressed in post-mitotic neurons [28, 29]. However, its expression has been demonstrated in almost all embryonic lateral glial cells and some NBs. In addition, elav-GAL4 drivers (458: elav^C155-GAL4, 8765: elav.L2-GAL4), which were not used in this study, modulate the expression of the reporter gene in embryonic glial cells and mitotically active cells [30]. Furthermore, the expression patterns of elav-GAL4 drivers differ between elav^C155-GAL4 (458) and other drivers (8760, 8765) [31]. Therefore, we monitored the mCD8-GFP reporter to investigate the expression pattern of elav-GAL4 (8760) in the larval CNS, particularly in the brain lobe. elav-GAL4 induced the expression of the reporter in cells expressing the Ani or NB marker Dpn, especially on the dorsal side of the larval brain lobe (Fig. S5). These results suggested that ani was knocked down in the NB with the elav-GAL4 driver. Therefore, we examined the distribution patterns of neurons and glial cells in the larval CNS of ani knockdown flies. The distribution patterns of neurons and glial cells in the CNS, especially in the elav > ani-IR⁵³³⁵⁸ larvae, were drastically perturbed, and the size of the brain lobe markedly increased (Figs. 5A, B). In addition to defects in the distribution patterns of neurons and glial cells, the size of the cells was drastically enlarged in the elav > ani-IR⁵³³⁵⁸ larvae (Fig. 5C). Similarly, the size of the whole CNS was drastically enlarged (Figs. 3C, 5A). However, the CNS shape was similar to that of the control. In addition, the ani knockdown larvae could crawl, although their crawling speed was significantly slower than that of the control larvae (Figs. 3E, F).

ani knockdown in the CNS affects the morphology of the NMJ and expression patterns of CadN in the NMJ

ani knockdown larvae showed locomotor defects. Therefore, we investigated whether motor neurons in the ani knockdown larvae had defects by examining the morphology of the NMJ, a specialized synapse between the nerve terminals of motor neurons and muscles. A detailed inspection of the NMJ revealed a substantial increase in the total branch length of synapses, the number of synaptic branches, and mature bouton numbers. In contrast, bouton sizes considerably decreased in elav > ani-IR⁵³³⁵⁸ larvae (Fig. 6). Although the effect was weaker than that in elav > ani-IR⁵³³⁵⁸ larvae, elav > ani-IR³⁴³⁴⁶⁵ larvae also showed a statistically significant increase in the number of synaptic branches and a decrease in bouton size (Fig. 6). Ani, an F-actin-binding protein, contributes to cytoskeleton formation at the edge of cells during neuronal migration and the strength of epithelial cell-cell adhesion [7, 11]. In addition, F-actin binds to catenin and cadherin, regulating adhesion between pre-and post-synapsis [32, 33]. Therefore, we hypothesized that ani contributed to the strength of adhesion between pre- and post-synaptic cells. As synaptic cell adhesion molecules, such as CadN, Capricious, Dscam, and Fasciclin II (Fas II), play important roles in the connection between pre-and post-synaptic cells, we evaluated the expression pattern of CadN in the NMJ of ani knockdown larvae. In the synaptic boutons in the NMJ of both lines of ani knockdown larvae, the size of the CadN foci and the total intensity of CadN significantly decreased whereas the number of CadN foci did not change (Fig. 7). These results suggest that CadN levels were decreased and that CadN clustering was impaired in the NMJ of the ani knockdown larvae.

Ani is expressed in almost all Dpn-expressing type I and II NBs and in two layers of Dac-expressing cells in the neuroepithelium posterior to the LF [35] (Fig. 1D). This is similar to the distribution pattern of proliferating cells in the brain lobe (Figs. 1D, S1). Ani is mainly localized to the nucleus during interphase [36–40] and to the cortex during the cytokinesis of proliferating cells [36, 37, 41]. Moreover, it is diffusively localized to the cytoplasm of non-proliferating cells [7, 42]. In the present study, Ani was strongly expressed in proliferating cells in the brain lobe and the thoracic region in the VNC (Figs. 1, 2, S1). Contrastingly, it was weakly expressed in some cells of the VNC, the axon, and the NMJ in the differentiated motor neurons (Figs. 2, S3). Ani was localized to the boutons in the NMJ (Fig. 2C). As actin is not required to expand membrane blebbing, it must be stabilized or retracted [43–45], suggesting that the bouton to which Ani is localized is robust and stabilized. Although it was predicted that decreased Ani at the bouton results in reduced mature boutons, the number of boutons contrarily increased with NMJ elongation in ani knockdown larvae (Fig. 6). However, the mean size of the boutons considerably decreased, which may suggest destabilization of the bouton structure. The increased NMJ length in ani knockdown larvae may be caused by a different mechanism. TGFβ/BMP and Wnt/Wg signaling are necessary for NMJ development. Null mutants of the highwire gene encoding an E3 ubiquitin ligase show a decrease in MAPKKK dial leucine zipper-containing kinase (DLK, called Wallenda in Drosophila) levels and synaptic overgrowth [46–48]. In addition, the loss-of-function allele of the short stop (shot) encoding a member of the spectraplakin family of large cytoskeletal linker molecules that bind microtubules and actin shows substantial overgrowth of the NMJ [48]. shot negatively regulates the Wallenda/DLK involved in the axonal injury response [48]. Because Ani stabilizes the F-actin network [34, 49], the elongation of NMJ following decreased ani levels may have been caused by a disturbance in TGFβ/BMP or Wnt/Wg signaling. Moreover, ani knockdown neurons may not sense the extracellular signal properly because the Ani reduction perturbed the distribution of CadN at the boutons in the NMJ. Although mutations and overexpression of the gene that induces NMJ overgrowth have been reported [49, 50], the electrophysiological function of the neuron was not or only slightly disordered. In addition, the larval locomotor ability or behavior was not evaluated.

The Ani size detected through Western blotting is usually larger than the predicted size calculated from the amino acid sequences [5, 37, 51], which is considered that the larger size is caused by an elongated coiled coil with low electrophoretic mobility. Consistent with previous results, Western blotting revealed an Ani size of approximately 178 kDa, which was larger than the 132.9 kDa calculated from the amino acid sequences in the present study. In addition, Ani dots connected by filament-like structures were detected in the axons of motor neurons (Fig. 2D). Furthermore, ani knockdown larvae, these Ani signals connected by filament-like structures were hardly detectable, and the morphology of the actin filaments appeared abnormal in the axons (Fig. S3). These data suggest the Ani forms a dimer and the dimerized Ani has a role in the actin filament assembly in the axon. However, no study has directly revealed that this protein dimerizes to date. However, further experiments are needed to confirm this result.

This study revealed the numerous roles of Ani in the nervous system of Drosophila, and the distribution patterns of Ani in the NMJ suggested its important role in nerve-muscle communication.

Fly stocks

Flies were cultured on standard food containing 0.65% agar, 10% glucose, 4% dry yeast, 5% corn flour, and 3% rice bran under a 12:12 h light-dark cycle at 25°C. Fly strains UAS-GFP.nls (107870), UAS-mCD8::GFP (108068), y¹v¹ (101249) were provided by the Kyoto Stock Center (Drosophila Genomics and Genetic Resources (DGGR)). Fly strains elav-GAL4 (8760), OK371-GAL4 (26160), UAS-ani-IR (53358), and UAS-LifeAct-GFP (35544) were purchased from the Bloomington Drosophila Stock Center (BDSC), BDSC. The UAS-ani-IR (33465) strain was obtained from the VDRC.

Crawling assay

The crawling assay was performed as previously described [52], with some modifications. Male larvae at the wander third instar stage were randomly collected and placed on 2% agar Petri dishes. The larval movements were recorded for 1 min using a digital camera. The recorded videos were analyzed using ImageJ 1.53t [53] and the wrMTrck plugin (developed by Dr. Jesper Søndergaard Pedersen, http://www.phage.dk/plugins/wrmtrck.html) to obtain the larval average speed [54].

Climbing assay

The climbing assay was performed as previously described [55]. Newly eclosed adult male flies of each strain were collected into separate vials at a maximum density of 20 flies/vial. The flies were maintained at 28°C under a 12:12 h light-dark cycle. Flies were transferred to cylindrical tubes with a 15 cm height and 2 cm diameter to measure their climbing ability. The cylinders were tapped five times to collect the flies at the bottom, and their movements were recorded using a digital camera. The procedure was repeated five times at 1 min intervals. In all experiments, the height to which each fly climbed after 5 s was scored from 0 to 5 points at 2 cm intervals from the bottom to calculate the climbing index. The assay was performed at 3, 7, and 14 days after eclosion.

Survival assay

Newly eclosed adult male flies were randomly placed in food vials at a maximum density of 20 flies/vial. They were subsequently transferred to a new vial every 2–3 days, and the number of dead flies was counted simultaneously [56].

Immunostaining

Larvae and adult flies were dissected in cold Phosphate Buffered Saline (PBS) and fixed with 4% paraformaldehyde (PFA) in PBS at 25°C for 20 min or 40 min for the brain lobe or NMJs, respectively. Next, they were washed with 0.3% PBS-T (PBS with 0.3% Triton X-100), blocked with 10% normal goat serum in 0.15% PBS-T, and incubated with the following primary antibodies: Anti-Ani antibody (rabbit, 1:400; kindly provided by Dr. Maria G. Giansanti [39, 57]), anti-Dlg1 IgG (mouse, 1:200, 4F3; Developmental Studies hybridoma Bank (DSHB)), anti-Dpn IgG (rat, 1:100, 11D1BC7; Abcam, Cambridge, UK), anti-Elav IgG (mouse, 1:50, 9F8A9; DSHB), anti-Elav IgG (rat, 1:50, 7E8A10; DSHB), anti-Lamin IgG (mouse, 1:100, ADL67.10; DSHB), Alexa 647 conjugated anti-HRP IgG (goat, 1:200; Jackson ImmunoResearch, West Grove, PA, USA), FITC conjugated anti-HRP IgG (goat, 1:200; Jackson ImmunoResearch), anti-CadN IgG (rat, 1:80, DN-Ex #8; DSHB). The samples were then washed with 0.3% PBS-T and incubated with a secondary antibody at 25°C for 2 h. Finally, they were washed with 0.3% PBS-T and mounted on Vectashield mounting media (Vector Laboratories, Newark, CA, USA). The samples were imaged using a confocal microscope (FLUOVIEW FV10i, Olympus, Tokyo, Japan). The collected images were analyzed using ImageJ 1.49o software.

BrdU labeling

CNS dissected from third instar larvae were incubated in 75 µg/µL Bromodeoxyuridine 5-bromo-2’-deoxyuridine (BrdU) at 30°C for 2 h. They were subsequently fixed with 4% PFA at 25°C for 20 min and washed with 0.3% PBS-T. Next, the cells were incubated with anti-Ani IgG at 4°C for 16 h, incubated with 3M HCl for 30 min, and rinsed once with PBS. Incorporated BrdU was visualized using anti-BrdU IgG (1:10) and washed with 0.3% PBS-T. The samples were then incubated with the secondary antibodies at 25°C for 2 h, washed PBS-T 0.3%, and mounted with Vectashield mounting media. The samples were imaged using a confocal microscope (FLUOVIEW FV10i). The images were analyzed using ImageJ 1.53t software.

RNA isolation and quantitative RT-PCR

Total RNA was isolated from 20 larval CNS samples using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Next, cDNA was synthesized using the PrimeScript RT reagent kit (TaKaRa, Kusatsu, Shiga, Japan). Quantitative RT-PCR (qRT-PCR) was subsequently performed using SYBR Premix Ex Taq II (TaKaRa) in the CFX96 Touch Real-Time PCR Detection System (BIO-RAD, Hercules, CA, USA) using a standard curve-based method calculated using the CFX Manager software (BIO-RAD). The following primers were used for qRT-PCR: GAPDH [58]: (fw 5′-GGAGCCACCTATGACGAAATC-3′ /rev 5′-TCGAACACAGACGAATGGG-3′), ani SV-A&B (fw 5′- ACTTCAGGATCACGCTAGAAATC − 3′ / rev 5′- GTCTTGATGCCACCCTTCTT-3′), ani SV-B & C (fw 5′- CCTGCCAAATACGACAAAGTG − 3′ / rev 5′- CACGACGAAATACCGAAATTG − 3′), ani SV-C (fw 5′- CTGCTTGCTATTTCTGCTCTG − 3′ / rev 5′- TTTATTGTTGGTCTTGTCTAGGG − 3′), ani all SV (fw 5′- TCACTATACGGCGGTAAACAAG-3′ / rev 5′- CTCCTGCTCCTGAATCTCAAC-3′)

Western blotting

Twenty larvae were dissected in cold PBS and kept at -80°C to collect the CNS. The CNS samples were homogenized in 2× sample buffer, denatured at 95°C for 5 min, separated using 8% tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Tricine SDS-PAGE), and transferred to PVDF membranes. Next, the samples were incubated with anti-Ani (1:4000) or anti-α-Tubulin (α-Tub) IgG (mouse, 1:8000, Sigma-Aldrich) as a primary antibody, followed by incubation with HRP-conjugated anti-rabbit (goat, 1:4000, Thermo Fisher Scientific, Waltham, MA, USA) or mouse IgG (goat, 1:10000, Thermo Fisher Scientific, Waltham, MA, USA) as a secondary antibody. Proteins were subsequently detected with the ECL SELECT reagent (Cytiva, Tokyo, Japan) and captured and analyzed using AE-9300H Ez-Capture MG (ATTO, Tokyo, Japan)). The specificity of anti-Ani IgG has been demonstrated by Giansanti [39, 57].

Imaging analysis and deconvolution

The immunofluorescence-stained samples were imaged using a confocal microscope (FLUOVIEW FV10i). The images were analyzed using ImageJ 1.53t software. The morphology of the NMJs was analyzed based on the parameters described by Nijhof et al. [59]. Individual data were averaged from four NMJs in Muscle 4 at segments A3 and A4. The point-spread function for deconvolution was generated using fluorescent beads from the PS-Speck™ Microscope Point Source Kit (Invitrogen, Carlsbad, CA, USA). The input image has a voxel size (height × width × depth = 0.02 × 0.02 × 0.15 µm). Deconvolution was performed using the Richardson-Lucy deconvolution method, the plugin CLIJx [60], and ImageJ 1.53t software.

Data analysis

All data were analyzed using GraphPad Prism version 10 for Windows (GraphPad Software, Boston, MA, USA). The sample size and statistical comparisons performed are described in the protocol of each experiment.

ACKNOWLEDGEMENTS

We thank Maria G. Giansanti for kindly providing anti-Ani IgG, and the DGGR, BDSC, and VDRC for fly strains.

COMPETING INTERESTS

The authors declare no competing or financial interests.

AUTHOR CONTRIBUTIONS

H.M.A. and H.Y. and designed the project, wrote the main manuscript text, prepared all figures, performed experiments, and analyzed the results. T.T.P.D provided the advice for the experiments and data analysis. All authors reviewed the manuscript.

FUNDING

This research was partially supported by the JSPS Core-to-Core Program, Asia-Africa Science Platforms (1202987), to H.Y.

DATA AVAILABILITY

All raw data used during the current study are available from the corresponding author on request.

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No competing interests reported.

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The roles of anillin in the Drosophila nervous system

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