Maintenance of cell type-specific connectivity and circuit function requires Tao kinase

Sensory circuits are typically established during early development, yet how circuit specificity and function are maintained during organismal growth has not been elucidated. To gain insight we quantitatively investigated synaptic growth and connectivity in the Drosophila nociceptive network during larval development. We show that connectivity between primary nociceptors and their downstream neurons scales with animal size. We further identified the conserved Ste20-like kinase Tao as a negative regulator of synaptic growth required for maintenance of circuit specificity and connectivity. Loss of Tao kinase resulted in exuberant postsynaptic specializations and aberrant connectivity during larval growth. Using functional imaging and behavioral analysis we show that loss of Tao-induced ectopic synapses with inappropriate partner neurons are functional and alter behavioral responses in a connection-specific manner. Our data show that fine-tuning of synaptic growth by Tao kinase is required for maintaining specificity and behavioral output of the neuronal network during animal growth.


Introduction
The function of a neuronal circuit is determined by synaptic strength and patterns of connectivity that allow information to flow in a specific manner to elicit behavior. Many circuits are formed during early development and undergo plastic changes including pruning and activity dependent refinement to establish and adjust functional connectivity [1][2][3][4][5] . While the mechanisms of circuit establishment and refinement have been extensively studied in many systems, a less well understood process is how circuits remain functional while the animal and nervous system are still growing. This process requires scaling growth, adjustment of synaptic strength or both to maintain functional output despite changes in input resistance due to larger dendritic trees or muscles. In principal, circuit output in a growing animal could be maintained by homeostatic control of neurotransmitter release, postsynaptic receptor expression or by addition of synapses. While the former have been studied extensively by challenging synaptic function 2 , the molecular mechanisms of how neuronal networks scale proportionally during animal growth and maintain their specificity and behavioral output are not well understood.
Drosophila larvae are an excellent system to study growth-related adjustments of circuit anatomy and function: the animals dramatically increase in size and enlarge their body surface 100-fold while maintaining structural and functional connectivity of their approximately 10,000 neurons [6][7][8] . Both, the peripheral and central nervous system anatomically scale with animal growth: prominently, sensory dendrites of larval dendritic arborization (da) neurons cover the entire body wall and scale with the animal to maintain coverage 9,10 . Similarly, synapse numbers and firing properties of motor neurons at the neuromuscular junction (NMJ) adjust during larval growth to maintain functional output [11][12][13][14] . In the central nervous system (CNS), motor neuron dendrites proportionally increase their size during larval growth while maintaining the overall shape and receptive field domain 8 .
Similar to the pioneering work on the C. elegans connectome, recent efforts to map Drosophila larval connectivity have now provided insight into circuit architecture and function of a more complex connectome [15][16][17][18][19] . This includes the nociceptive class IV da (C4da) sensory neurons, which connect to an extensive downstream network and mediate responses to noxious mechanical and thermal stimulation, resulting in stereotyped rolling escape behavior [20][21][22] . Recent electron microscopy (EM)-based reconstruction of the C4da neuron 2 nd order network revealed at least 13 subtypes consisting of 5 different local, 3 regional, 1 descending and 4 ascending classes of interneurons 6 . In addition, this study has established that topography and sensory input are preserved in the early and late stage larval brain suggesting anatomical and functional scaling of the nociceptive network. Indeed, most larval behaviors including nociceptive responses are conserved throughout all stages suggesting that the majority of larval circuits maintain their function during animal growth 23 . In recent years, a subset of C4da 2 nd order neurons has been studied in greater detail including A08n, DnB, Basin and mCSI neurons, which have been shown to be sufficient for nociceptive rolling behavior when activated by opto-or thermogenetic means 18,[24][25][26][27][28] .
Functional network analyses by these and additional studies have revealed a hierarchical network organization, multisensory integration, and modality and positionspecific network functions suggesting extensive processing and modulation of nociceptive inputs 18,24,29 . This system thus offers a unique opportunity to probe how CNS circuit growth is regulated while preserving specific connectivity and functional output.
We and others have previously characterized A08n interneurons, which are major postsynaptic partners of C4da neurons required for nociceptive behavior 24,27,28 . Here we characterize the developmental changes of the C4da-A08n circuit during larval growth at the synaptic level. We show that the number of pre-and postsynaptic sites as well as connectivity is proportionally increasing during larval development. We identified the conserved Ste20-like kinase Tao as a negative regulator of postsynaptic growth in A08n neurons. Loss of Tao function induces aberrant growth of dendrites and increased numbers of postsynaptic specializations. Strikingly, a subset of A08n postsynapses were no longer confined to the C4da presynaptic domain, but formed synapses with sensory neurons innervating adjacent regions of the neuropil. We show that these ectopic synapses are functional and result in altered network output and behavior. Our findings suggest that Tao kinase is required for maintenance of specific connectivity and function during animal growth by restricting postsynaptic growth in a circuit specific manner.

Quantitative analysis of C4da and A08n neuron synapses
To evaluate the extent of synapses formed by neurons in the larval nociceptive circuit, we focused our efforts on establishing methods to visualize and quantify connections between C4da and A08n neurons, which display extensive synaptic contact along the entire ventral nerve cord (VNC) 24 . To this end, we used three independent methods to assess synaptic connectivity by i) employing synapse-specific GFP reconstitution across synaptic partners (Syb-GRASP 30 ), ii) measuring the apposition of pre-and postsynaptic marker proteins 31 and iii) performing immuno-electron microscopy (EM) of synaptic markers labeling C4da-A08n neuron synapses 24 . We first quantified the number of synaptic GRASP puncta from C4da-A08n neuron synapses in 3 rd instar larvae at 96 h after egg laying (AEL) using blind analysis of deconvolved 3D image stacks with automatic thresholding of synaptic puncta (details in methods). We consistently detected an average of 70-80 Syb-GRASP puncta per hemisegment ( Fig.   1A-C, F).
To facilitate comparison of GRASP synapse numbers with C4da and A08n neuron synaptic sites, we used the active zone marker Brp short -mCherry 32 to label C4da neuron-specific presynapses. In order to label A08n postsynaptic densities, we used Drep2-GFP previously shown to discretely label postsynaptic densities when expressed in mushroom body Kenyon cells 33 (Fig. 1D,E). We detected close apposition of Brp short -mCherry and Drep2-GFP at discrete foci in areas of C4da-A08n contact, and analyzed the number of co-localized C4da-A08n neuron synaptic puncta using automatic thresholding of apposed Brp/Drep2 puncta together with a distance threshold similar to previous work 31,34 (Fig. 1F, Supplementary Figure 1A-C, see methods for details). Synapse numbers determined using this approach were comparable to numbers from our Syb-GRASP analysis, suggesting that both methods allowed us to estimate C4da-A08n neuron connectivity. We further analyzed the number of C4da presynaptic and A08n postsynaptic puncta in different abdominal segments: overall numbers were similar from segment to segment, with C4da neurons displaying about 2-3-fold higher presynaptic counts compared to A08 postsynapses Lastly, we performed immuno-EM labeling of C4da-A08n connectivity in larvae expressing Brp short -mCherry (C4da) and Drep2-GFP (A08n). We first counted the total number of synapses on C4da and A08n neurons and then calculated the percentage of synapses that contained A08n postsynapses and C4da presynapses, respectively ( Fig. 1H, Supplementary Figure 1F). We found that ~30% of C4da neuron active zones formed synapses with A08n neurons and that ~50% of Drep2-GFP labeled A08n postsynaptic sites contacted C4da presynapses. Strikingly, the relative C4da-A08n synapse numbers we observed using EM and light microscopy were indistinguishable.
Taken together, our analysis shows that light microscopic pre-and postsynaptic marker apposition and Syb-GRASP analysis provide valid representations of C4da-A08n neuron connectivity.

C4da-A08n neuron connectivity scales with larval growth
We next wanted to assess C4da and A08n neuron connectivity across larval development. Drosophila larvae grow extensively after hatching and dendrite length and synaptic numbers at the NMJ and in the CNS have been shown to increase for the subsets of neurons investigated so far 6,8,10,34,35 . We analyzed C4da-A08n neuron synapse numbers from 48 h to 120 h AEL using Syb-GRASP and synaptic marker apposition ( Fig. 2A,B). Both methods showed a comparable linear increase of synaptic numbers from 48 h to 96 h AEL, with synapse numbers close to doubling every 24 h.
We observed a decline of C4da-A08n synapses at 120 h AEL using Syb-GRASP but not colocalization analysis, hinting at potential changes in their connectivity in wandering stage larvae ( Fig. 2A). C4da neuron presynaptic puncta kept increasing until 120 h, while A08n postsynaptic counts plateaued at 96 h (Fig. 2C,D). We then calculated the ratio of C4da-A08n neuron connections across development and found that the relative C4da presynaptic output to A08n neurons displayed mild alterations between the analyzed developmental timepoints, but remained within a range between 20-30% (Fig. 2E). In contrast, we observed a significant increase in synapse/postsynapse ratios for A08n neurons from 48 h to 72 h AEL indicating a developmental increase in their relative connectivity to C4da neurons during the transition from 2 nd to 3 rd instar stages (Fig. 2E). Taken together, these data show that C4da-A08n neuron synaptic numbers scale with larval growth and undergo stagespecific adjustments in connectivity.

Tao kinase function restricts postsynaptic growth of A08n neurons
We next focused on how A08n postsynaptic growth might control synaptogenesis with C4da neurons. In a candidate RNAi screen for growth-related genes we identified Tao kinase as a regulator of synaptic growth in A08n neurons. We perturbed Tao function in A08n or C4da neurons using RNAi-mediated knockdown (Tao RNAi ) or by overexpression of a hyperactive form of Tao (Tao CA ) 36 and analyzed synapse numbers using our newly established methods. A08n-specific knockdown of Tao resulted in a significant increase of A08n postsynaptic puncta at 96 h AEL (Fig. 3A,B'). In contrast, Tao hyperactivation caused a reduction of Drep2-GFP puncta. A08n neuron expression of Tao RNAi did not significantly affect C4da presynaptic or C4da-A08n synaptic numbers, while Tao CA overexpression strongly reduced both, suggesting that hyperactivation of Tao function negatively regulates C4da-A08n neuron synaptic connectivity (Fig. 3A, B-B´´). We sought to validate these results using Syb-GRASP and found that while Tao RNAi in A08n neurons did not affect C4da-A08n synapse numbers, Tao CA expression reduced GRASP puncta to a comparable extent as observed by our co-localization analysis (Fig. 3C,D). We also tested if Tao kinase was involved in presynaptic control of C4da-A08n neuron connectivity. Interestingly, C4da neuron-specific Tao RNAi expression did not affect synaptic marker numbers at 96 h AEL, while Tao CA overexpression strongly reduced C4da pre-, A08n post-and C4da-A08n synaptic numbers (Supplementary Figure 2A We next examined the developmental profile of ectopic postsynaptic puncta of A08n neurons, which were not localized within the C4da neuron presynaptic domain upon loss of Tao function. We therefore analyzed the number of postsynaptic Drep2-GFP puncta that overlapped with the C2da/C3da presynaptic domain labeled by anti-Fas3 immunostaining (Fig. 4F,G,G'). In controls, we rarely observed A08n postsynapses localized outside of the C4da neuron presynaptic domain. In contrast, Tao RNAi in A08n neurons led to ectopic A08n postsynapses that were displaced laterally within the adjacent domain of C2da/C3da sensory neuron projections. Ectopic A08n postsynapses were already present at 48 h AEL and persisted to a similar degree throughout development (Fig. 4F). This suggests that Tao kinase function is required to prevent ectopic postsynaptic sites by restricting the A08n postsynaptic domain.

Conserved Tao kinase activity regulates postsynaptic growth
Overexpression of hyperactive Tao kinase resulted in a strong decrease of A08n Drep2-GFP puncta (see Fig. 3), which might indicate kinase activity-dependent regulation of postsynaptic growth in A08n neurons. To test this hypothesis further and to probe potentially conserved Tao activity, we asked if the closest human orthologue, Tao kinase 2 (hTaoK2), was capable of compensating for the loss of Drosophila Tao.
TaoK2 has recently been shown to affect dendritic and synaptic development in mammals and has been linked to Autism spectrum disorders (ASDs) based on patient mutations that alter its kinase activity 39-41 . We compared the ability of hTaoK2 or a kinase activity-impaired ASD-linked variant (hTaoK2 A135P ) to rescue loss of Tao in We also tested if loss of Tao affects functional connectivity of C4da and A08n neurons.
Using optogenetic activation of C4da neurons, we detected a significant decrease in A08n neuron responses after loss of Tao compared to controls (Supplementary Figure   6B-D). These data show that loss of Tao in A08n neurons gives rise to functional ectopic connectivity with C3da sensory neurons while partially impairing C4da-A08n neuron physiological output.

Ectopic Tao-dependent connectivity alters somatosensory network function and behavioral action selection
To dissect the impact of Tao-dependent connectivity changes, we analyzed behavioral consequences of Tao loss of function in this system. We focused on C3da, cho and To address if Tao-dependent ectopic C3da-A08n neuron connectivity contributed to mechanonociceptive behavior, we expressed Tetanus toxin light chain (TNT) in C4da neurons while reducing Tao function in A08n neurons. Interestingly, nociceptive behavior was partially restored compared to TNT expression alone, possibly due to functional activation of A08n neurons by C3da neurons (Fig. 7B). In contrast, C4da neuron specific optogenetic activation revealed that A08n neuron output is required for Tao-dependent behavioral changes, as simultaneous silencing blocked any differences (Supplementary Figure 7D). These findings suggest a direct contribution of loss of Tao-induced C3da-A08n neuron connections to mechanonociceptive behavior.
Lastly, we examined whether ectopic connectivity between C3da and A08n neurons could affect noxious cold responses, which are mediated by C3da/cho neurons 45,47 .
We first assayed if A08n neurons could respond to a cold stimulus after loss of Tao.
To this end, we analyzed calcium-dependent photoconversion of the calcium integrator CaMPARI 49 in A08n neurons in response to noxious cold. We observed a significant increase in CaMPARI photoconversion in Tao RNAi -expressing A08n neurons following a noxious cold stimulus, while controls displayed only low level responses (Fig. 7C).
Next we addressed if this altered activation of A08n neurons could affect noxious cold behavior using a cold plate assay. Freely crawling larvae (96h AEL) were cooled from 25 °C to 3 °C within 50 seconds, which resulted in sequential stop & turn behavior  Figure 7E). Taken together, these data show that loss of Tao-induced connectivity changes between C3da and A08n neurons functionally alter noxious C3da/cho-dependent cold and C4da-dependent mechanonociceptive responses thus perturbing modality-specific behavioral action selection.

Discussion
Evolution shaped precise neuronal networks that elicit appropriate sensory modalitydependent actions. To select for accurate behavioral responses or sequences, combinations of feedback or feedforward inhibition, disinhibition and co-activation are thought to result in command-like decisions selecting a certain behavior or induce probabilistic behavioral sequences 18,48,[50][51][52] . Yet organismal size increase during juvenile development also requires proportional growth of its nervous system to maintain connectivity and thus the appropriate behavioral output. For example, zebrafish larvae already display prey catch behavior 5 days post fertilization, which is maintained and refined until adulthood despite undergoing 5-fold growth in body length and further nervous system development 53 . Similarly, Drosophila larvae display preservation of most behaviors including nociceptive and mechanosensory responses from early to late stages 23  The observed proportional growth of receptive fields is required to maintain spacing and connectivity, yet the underlying molecular mechanisms have not been fully elucidated. Both, activity-dependent and -independent mechanisms, are known to contribute to circuit refinement and stability 5 . How either of these is required for network scaling and specificity during organismal growth remains to be elucidated in detail. Our results point to growth-related factors as major players in this process: Tao function restricts dendritic and synaptic growth during Drosophila larval development, which is critical for maintaining circuit specificity. Tao prevents dendritic overgrowth in a cell-autonomous fashion suggesting its activity is tightly controlled, possibly by extrinsic signals required to coordinate synaptic growth of partner neurons. This This suggests that the nociceptive circuit is subject to structural and functional plasticity responding to both growth and activity-related cues. As Tao kinase signaling has been linked to dendrite growth and cytoskeletal regulation 36,40,66,67 , it is likely that it controls the intracellular machinery required to coordinate actin and microtubule dynamics in response to extrinsic signals, thus regulating scaled dendritic and synaptic growth.
Our data show that Tao's role in proportional growth is likely conserved as its closest human orthologue Taok2 was able to substitute for Drosophila Tao function.
Interestingly, an ASD-linked kinase-impaired Taok2 variant 39 did not recover function suggesting that Tao kinase activity is essential for its role in growth regulation.

Drosophila melanogaster stocks
All stocks were maintained at 25 °C and 70% rel. humidity with a 12 h light/dark cycle on standard fly food unless noted otherwise. All transgenic lines were maintained in a white mutant (w -) or yellow white mutant (y -,w -) background. Stocks were obtained from the Bloomington (BDSC) Drosophila stock centers unless otherwise indicated.
We used the following lines:

Line
Labels Source

Immunohistochemistry and confocal microscopy
Larval brains were dissected in dissection buffer (108 mM NaCl, 5 mM KCl, 4 mM

Mechanonociception assays
Mechanonociception experiments were performed with calibrated von-Frey-filaments (35 mN or 50 mN) and staged 3 rd instar larvae (96h AEL ±3h) 23 All behavioral assays and analyses were performed in a blinded and randomized fashion.