Conserved Tao Kinase Activity Regulates Dendritic Arborization, Cytoskeletal Dynamics, and Sensory Function in Drosophila

Abstract

Dendritic arborization is highly regulated and requires tight control of dendritic growth, branching, cytoskeletal dynamics, and ion channel expression to ensure proper function. Abnormal dendritic development can result in altered network connectivity, which has been linked to neurodevelopmental disorders, including autism spectrum disorders (ASDs). How neuronal growth control programs tune dendritic arborization to ensure function is still not fully understood. Using Drosophila dendritic arborization (da) neurons as a model, we identified the conserved Ste20-like kinase Tao as a negative regulator of dendritic arborization. We show that Tao kinase activity regulates cytoskeletal dynamics and sensory channel localization required for proper sensory function in both male and female flies. We further provide evidence for functional conservation of Tao kinase, showing that its ASD-linked human ortholog, Tao kinase 2 (Taok2), could replace Drosophila Tao and rescue dendritic branching, dynamic microtubule alterations, and behavioral defects. However, several ASD-linked Taok2 variants displayed impaired rescue activity, suggesting that Tao/Taok2 mutations can disrupt sensory neuron development and function. Consistently, we show that Tao kinase activity is required in developing and as well as adult stages for maintaining normal dendritic arborization and sensory function to regulate escape and social behavior. Our data suggest an important role for Tao kinase signaling in cytoskeletal organization to maintain proper dendritic arborization and sensory function, providing a strong link between developmental sensory aberrations and behavioral abnormalities relevant for Taok2-dependent ASDs.SIGNIFICANCE STATEMENT Autism spectrum disorders (ASDs) are linked to abnormal dendritic arbors. However, the mechanisms of how dendritic arbors develop to promote functional and proper behavior are unclear. We identified Drosophila Tao kinase, the ortholog of the ASD risk gene Taok2, as a regulator of dendritic arborization in sensory neurons. We show that Tao kinase regulates cytoskeletal dynamics, controls sensory ion channel localization, and is required to maintain somatosensory function in vivo Interestingly, ASD-linked human Taok2 mutations rendered it nonfunctional, whereas its WT form could restore neuronal morphology and function in Drosophila lacking endogenous Tao. Our findings provide evidence for a conserved role of Tao kinase in dendritic development and function of sensory neurons, suggesting that aberrant sensory function might be a common feature of ASDs.

Keywords: Tao kinase; autism spectrum disorders; cytoskeletal dynamics; dendritic arborization; sensory neuron.

Figure 1.

Tao is a negative regulator of dendritic arborization in da sensory neurons. A, Schematic diagram of the Tao exon-intron structure, protein domain organization, Tao mutant alleles, and transgenic constructs (N/C: N/C terminus, point mutations, deletion gaps, and domain distribution; full-length: WT Tao; Tao^eta: Tao-null mutant; D168A: kinase-inactive form; Δ423-900: hyperactive form). B, MARCM analysis of dendritic phenotypes of Tao-null mutant C4da neurons without and with overexpression of different transgenic Tao constructs as indicated. Scale bar, 100 μm. C–E, Analysis of total dendrite length (C), number of terminals (D), and dendritic complexity by Sholl analysis (mean ± SD) (E) of Tao^eta mutant C4da neurons (n = 5, n = 5, n = 7, n = 5, n = 5). *p < 0.05, ***p < 0.001 compared with Ctl (one-way ANOVA with Bonferroni post hoc test). F, MARCM analysis of control (Ctl) and Tao^eta mutant C1da neurons. G–I, Analysis of total dendrite length (G), number of terminals (H), and dendritic complexity by Sholl analysis (I) of Tao^eta mutant C1da neurons (n = 5/genotype). ***p < 0.001 compared with Ctl (two-tailed unpaired Student's t test).

Figure 2.

Tao controls dendritic arborization of da neurons in a kinase activity-dependent manner and is endogenously expressed in da sensory neurons. A, Representative images of dendritic phenotypes after manipulation of Tao activity in C4da neurons (ppk-Gal4, UAS-CD4-tdGFP). Scale bar, 100 μm. B–D, Quantitative analysis of dendritic length (B), terminal numbers (C), and dendrite complexity by Sholl analysis (D) of the indicated genotypes (n = 10/genotype). **p < 0.01, ***p < 0.001 compared with Ctl (one-way ANOVA with Bonferroni post hoc test). E–G, Analogous analysis of C1da neuron morphology after Tao kinase perturbation (98B-Gal4, UAS-CD4-tdGFP) with quantification of dendritic length (F) and terminal numbers (G) (n = 11, n = 14, n = 14, n = 13, n = 17). **p < 0.01, ***p < 0.001 compared with Ctl (one-way ANOVA with Bonferroni post hoc test). H, C4da neurons (ddaC) in third instar larvae visualized by Gal4^ppk-driven mCD4-tdGFP and anti-Tao immunostaining under control, Tao^RNAi, or Tao overexpression (Tao^FL) conditions. Purple arrows indicate somata. Yellow dotted lines indicate C4da soma. Scale bar, 10 μm. I, Relative fluorescence intensity of anti-Tao signals normalized to GFP (n = 13 neurons from >5 animals). **p < 0.01, ***p < 0.001 compared with Ctl (one-way ANOVA with Bonferroni post hoc test). J, Mean intensity of anti-Tao signal in C4da (ddaC) and C1da (ddaE) neurons (n = 11 for each genotype from at least 6 larvae). K, Endogenous anti-p-Tao immunostaining in third instar larval filets with C4da neurons labeled by CD4-tdGFP expression. Yellow dotted lines indicate C4da neuron soma and dendrites. K′, C4da neuron dendrite portion resliced in Z direction (as shown in D) and displaying discrete dendritically localized p-Tao puncta. Scale bar, 50 μm. L, Manipulation of Tao activity levels by Tao^RNAi or Tao overexpression in da neurons using 21–7-Gal4 and anti-p-Tao immunostaining. da neurons were visualized by anti-HRP. Tao^RNAi reduces p-Tao levels, whereas Tao overexpression increases p-Tao levels in da neurons. Scale bar, 20 μm. M, Quantitative analysis of p-Tao levels in da neuron somata normalized to HRP signals (n = 7/genotype). *p < 0.05, ***p < 0.001 compared with Ctl (one-way ANOVA with Bonferroni post hoc test).

Figure 3.

Tao developmentally limits dendritic arborization in da sensory neurons. A–D, Developmental analysis of C4da neurons dendritic morphogenesis with MARCM. A, Representative images showing dendritic development of the same Control (Ctl) and Tao^eta mutant C4da neuron from 48 to 96 h AEL. Green represents the new branches generated during different time windows. Scale bar, 100 μm. B, C, Quantitative comparison of dendritic length (B) and terminal numbers (C) of Ctl and Tao^eta C4da neurons from 48 to 96 h AEL. The Tao^eta phenotype was already visible at 48 h AEL (n = 5/genotype). *p < 0.05, **p < 0.01, ***p < 0.001 compared with Ctl (two-tailed unpaired Student's t test). D, The number of newly added terminals during the different time points are shown (n = 5/genotype). **p < 0.01 (two-tailed unpaired Student's t test). E–H, Developmental analysis of C1da neuron dendritic morphogenesis using Tao^RNAi. E, Representative images showing dendritic development of the same Control and Tao^RNAi C1da neurons from 48 to 96 h AEL. Red and blue circles represent new branches generated from 48–72 h and 72–96 h AEL, respectively. Green represents the new branches generated during different time windows. Scale bar, 50 μm. F, G, Quantitative comparison of dendritic length (F) and terminal numbers (G) of control and Tao^RNAi C1da neurons from 48 to 96 h AEL (n = 5/genotype). *p < 0.05, **p < 0.01, ***p < 0.001 compared with Ctl (two-tailed unpaired Student's t test). H, The number of newly added terminals during the different time points are shown (n = 5/genotype). *p < 0.05, ***p < 0.001 (one-way ANOVA with Bonferroni post hoc test).

Figure 4.

Tao affects dendritic terminal dynamics. A, Dendritic terminal dynamics of C4da ddaC neurons in third instar (∼96 h AEL) larvae were time-lapse imaged every 5 min over a 30 min interval. Representative images show dendritic terminal dynamics at 0 and 30 min in Ctl, Tao^RNAi, and Tao^FL C4da neurons. B, Quantification of terminal branch extension (green), retraction (red), and stable terminals (gray). C, Quantification of ratios between extension and retraction. D, Quantification of terminal branch extension (positive value) and retraction (negative value) speed revealed by time-lapse confocal images over 5 min intervals. Growth speed is reduced for Tao^RNAi and even more for Tao^FL expression. E, F, Cumulative frequency of growth speed for extension (E) and retraction (F) (n = 80, n = 61, n = 60 terminals from 4 neurons/genotype). **p < 0.01, ***p < 0.005 compared with Ctl (Mann–Whitney U test). Scale bar, 20 μm.

Figure 5.

Tao kinase activity regulates dendritic MT dynamics. A–J, MT dynamics in C4da neurons. A, Schematic image showing a C4da neuron at 96 h AEL. The main branches (red) and terminal dendrites (blue) are labeled. The proximal dendrites were defined as 0–160 μm (purple box), distal dendrites as 160–320 μm from the soma. B, Representative kymographs showing EB1-GFP comets in control or after manipulation of Tao kinase activity by specific overexpression of indicated Tao transgenes in C4da neurons (ppk-Gal4). Scale bar, 10 μm. C–E, Quantitative data showing the number (C), polarity (D), and velocity (E) of EB1-GFP comets in proximal or distal dendrites and overall comparison (n = 10/genotype). *p < 0.05, **p < 0.01, ***p < 0.001 compared with Ctl (one-way ANOVA with Welch test). F–J, For analysis of MT dynamics in terminal branches, only dendrites showing no additional higher order branches were defined as terminal dendrites. The percentage of terminals containing EB1 (F), the distribution of the number of comets observed per branch (G), the number of EB1 in each terminal branch (H), polarity of MTs in each terminal (I), and velocity of dynamic MTs (J) are shown. Exact p values are indicated (Student's t test). K–O, MT dynamics in C1da neurons. K, Representative kymograph showing EB1-GFP comets in control or after Tao^RNAi manipulation in C1da neurons. Magenta dots represent individual MT nucleation and growth events. Yellow dots represent two or more individual comets originating from a single nucleation site. Scale bar, 50 μm. L–O, Quantitative data showing the EB1 comet numbers (L), number of nucleation sites (M), polarity (N), and velocity (O) (n ≥ 15/genotype). **p < 0.01, ***p < 0.001 (unpaired Student's t test).

Figure 6.

Tao regulates Futsch/Map1b levels and distribution in C4da neurons. A, Comparison of Futsch-positive MTs in WT (Ctl), Tao^RNAi- and Tao^FL-expressing C4da da neurons. Endogenous Futsch (anti-Futsch (22c10): 1:100) is detected in all da neurons. The relative intensity of Futsch normalized to CD4::tdGFP expressed only in C4da neurons is shown. The high-intensity (red) dendrites are from non-C4da neurons. Scale bar, 50 μm. B, Quantification of relative Futsch intensities (n = 9, n = 10, n = 10). *p < 0.05 (one-way ANOVA with Bonferroni correction). C, Normalized Futsch signal along the longest primary dendrites was plotted against increasing soma distance showing the different intensity profiles of Ctl, Tao^RNAi, and Tao^FL C4da neurons. Solid lines indicate mean levels. Shaded area represents SD.

Figure 7.

Tao affects F-actin levels and distribution in C4da neurons. F-actin was visualized by Lifeact-GFP in C4da neurons labeled by CD4::tdTomato. A, Representative images of C4da neurons expressing Lifeact-GFP and relative intensity normalized to mCD4::tdTomato. B–D, Quantification of relative Lifeact-GFP intensity in (B) main branches, (C) terminals, and (D) terminal to main branch ratios. Scale bar, 50 μm. n ≥ 8. *p < 0.05, **p < 0.01, ***p < 0.001 compared with Ctl (one-way ANOVA with Bonferroni correction).

Figure 8.

Manipulation of Tao activity affects levels and distribution of mechanosensory channels. A, Endogenous ppk26 immunoreactivity (magenta) was detected on dendrites of C4da neurons by immunohistochemistry under nonpermeabilizing conditions in WT (Ctl) C4da neurons. Anti-HRP signal labels the cell surface of all sensory neurons and was used for normalization. Relative intensity (RI) of ppk26 normalized to the HRP signal is shown. Enlarged white (main branches) and orange (terminals) boxes show that ppk26 immunoreactivity along the dendrites is not uniform. B, The dendritic length from RI images was measured. The ppk26 coverage index was defined as the length of ppk26-positive dendrites divided by the corresponding total dendritic length. The ppk26 index in a single dendrite ranges from 0 (ppk26 RI signals could not be detected throughout the whole dendrite) to 1 (RI of ppk26 signals are detected throughout the whole dendrite). C, Quantifications comparing the ppk26 index of control (Ctl), Tao^RNAi, and Tao^FL in C4da neuron main branches. D, The overall RI of ppk26 in main branches was measured. E–G, Quantifications comparing the ppk26 index of Ctl, Tao^RNAi, and Tao^FL in distal terminal branches. E, The average ppk26 coverage index was shown. Each dot represents a terminal branch. F, Distribution analysis of binned ppk26 coverage indexes for the indicated genotypes. G, The RI of ppk26 in terminal branches was measured and plotted for the indicated genotypes. Scale bar, 50 μm. C, D, n > 40, 40, 35 main branches from 8, 8, 7 neurons. H, J, n > 100, 100, 70 terminals from 8, 8, 7 neurons. **p < 0.01, ***p < 0.001 (one-way ANOVA with Bonferroni correction). H, Endogenously GFP-tagged TrpA1 channel expression (+/TrpA1^{MI07645-GFSTF.0}) was detected by anti-GFP immunostaining in controls or with Tao^RNAi and Tao^FL expression in sensory neurons (21–7-Gal4). Anti-HRP immunoreactivity was used for normalization. TRPA1 is only expressed in C4da neurons. Scale bar, 20 μm. I, Statistical analysis of TrpA1 expression levels in somata of C4da neurons (n = 12/genotype). ***p < 0.001 comparing with Ctl (one-way ANOVA with Bonferroni correction).

Figure 9.

Human TaoK2, but not an ASD-linked variant, can functionally replace Drosophila Tao. A, Representative images showing dendritic morphology of C4da neurons after knockdown of Tao and rescue with hTaoK2 WT or A135P and A335V variants in C4da neurons. Scale bar, 100 μm. B–D, Quantitative analysis of the total dendritic length (B), number of terminals (C), and number of EB1 comets/dynamic MTs (D) in Tao^RNAi and Tao^RNAi/hTaoK2-expressing C4da neurons (n = 10/genotypes for A–C; n = 12/genotypes for D). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Bonferroni post hoc test). E, Schematic larval rolling response to mechanonociceptive stimulation using a 45 mN von Frey filament. E′, Mechanonociceptive rolling responses of third instar larvae (96 h AEL) after C4da neuron-specific knockdown of Tao and rescue with hTaoK2 WT, A135P, and A335V variants (n = 73, n = 70, n = 72, n = 70, n = 72). *p < 0.05, **p < 0.01, ***p < 0.001 (Kruskal–Wallis test with Dunn's post hoc).

Figure 10.

Developmental or adult-specific loss of Tao function affects dendritic arborization of adult sensory neurons. A–D, Developmental loss of Tao function results in dendritic overbranching in adult sensory C4da neurons. A, Dendritic morphology and traced dendrites of v'ada C4da neurons are shown. Red circles represent the soma. Scale bar, 50 μm. B–D, Quantification of dendritic length (B), number of terminals (C), and Sholl analysis (D) (n = 10/genotype). *p < 0.05, ***p < 0.001 (t test). E–H, Adult-specific loss of Tao function results in dendritic overbranching. E, Dendritic morphology of v'ada C4da neurons is shown with or without expression of Tao^RNAi at the restrictive (29°C) or permissive (18°C) temperature, respectively. Scale bar, 50 μm. F–H, Quantification of dendritic length (F), number of terminals (G), and Sholl analysis (H) (n = 10/genotype). *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA with Bonferroni correction).

Figure 11.

Tao activity in sensory neurons is required for adult motor reflexes and social interaction. A, Performance of Tao^η/+ (light gray bar) and control female flies (black bar) in negative geotaxis (n = 30, n = 19). Data are mean ± SE. ***p < 0.001 (Mann–Whitney test). B, Locomotor activity of Control and Tao^eta/+ flies was monitored over 24 h. Gray represents light-off periods. C, Quantification of activity during light ON and (D) light OFF period; n = 24, n = 55 (individual flies) for each genotype were tested. Data are mean ± SE. ****p < 0.0001 (Mann–Whitney test and t test). E, Control and Tao^eta/+ female flies were analyzed for total number of social interactions in vials (average of number of total approaches, lunges, tussles, wing threat, and initiation of courtship) during a period of 2 min; n = 12 independent experiments (each with 8 flies) for each genotype were analyzed. Data are mean ± SEM. ****p < 0.0001 (t test). F, Negative geotaxis and (G) social interaction upon Tao^RNAi expression specifically in somatosensory neurons (PNS) using Gal^5–40. F, n = 31, n = 26, n = 27, n = 31, n = 26. G, n = 16, n = 12, n = 12, n = 10, n = 10, n = 12 independent experiments (each with 8 flies)/genotype. Data are mean ± SE. *p < 0.05, **p < 0.01 (Kruskal–Wallis test with Dunn's post hoc). H, Social behavior after pan-neuronal RNAi-mediated knockdown of Tao throughout development or (I) in adults. Light gray bars represent genotypes with downregulated Tao. Black bars represent genetic control strains. Dark gray bars represent genetic rescue lines (UAS-TaoK2^WT/A135P, UAS-Tao^RNAi). H, n = 13, n = 13, n = 12, n = 9. G, n = 16, n = 13, n = 11, n = 10, n = 9, n = 10, n = 10, n = 10 independent experiments (each with 8 flies)/genotype. Data are mean ± SE. **p < 0.01, ***p < 0.001 (Kruskal–Wallis test with Dunn's post hoc).