Modulation of a critical period for motor development in Drosophila by BK potassium channels

Critical periods are windows of heightened plasticity occurring during neurodevelopment. Alterations in neural activity during these periods can cause long-lasting changes in the structure, connectivity


In brief
Lowe et al. show that a gain-of-function mutation in the BK potassium channel linked to movement disorders acts during a late stage of neural development to impair limb kinematics and locomotion in adult fruit flies.They further show that BK channel gain of function disrupts neurotransmission and synaptic maturation during this critical period.

INTRODUCTION
During development, neural networks within vertebrate nervous systems become electrically excitable and exhibit stimulus-independent bouts of excitatory activity as well as responses to sensory inputs. 1 Calcium (Ca 2+ )-dependent signaling pathways occurring downstream of such activity influence intrinsic excitability and synaptic maturation in developing neurons. 2 Perturbations in this process can thus lead to long-lasting changes in neural connectivity and robustness, particularly during windows of heightened plasticity termed critical periods. 3ritical periods have been extensively studied in the context of the visual system, with classic work showing that monocular eye closure during development, but not adulthood, leads to lasting changes in neural connectivity in the visual cortex. 4Critical periods have also been documented for motor control, 5 and stimulus-independent neural activity has been detected in developing motor regions such as the cerebellum and the spinal cord. 1,6Suppressing this activity results in a variety of defects in pre-motor circuit development and movement, including axonal mis-wiring, 7 delayed onset of coordinated locomotion, 8 and aberrant patterns of muscle activation. 9However, endogenous genetic regulators of critical periods influencing motor development remain poorly defined.
Big K + (BK) channels represent an intriguing candidate modifier of critical periods relating to movement.BK channels bind Ca 2+ via intracellular C-terminal domains and couple increases in intracellular Ca 2+ to K + efflux across membranes. 10Such ion flux may influence neuronal physiology and plasticity through several mechanisms.For example, axonal and presynaptic BK channels drive action potential (AP) repolarization 11 and suppress presynaptic neurotransmitter release, respectively, 12 while dendritic BK channels impact spike-timing-dependent plasticity by negatively tuning NMDA receptor (NMDAR)-mediated Ca 2+ currents. 13Activity-dependent changes in alternative splicing of BK channel mRNA also drives homeostatic alterations in AP duration in response to chronic inactivity. 14Because BK channels are expressed in the developing vertebrate brain, 15,16 these channels are poised to influence both neural activity and plasticity during development and, thus, the maturation of motor circuits.
Intriguingly, inherited and de novo gain-of-function (GOF) mutations in the BK channel a-subunit (BK GOF) cause dystonia and dyskinesia in humans, [17][18][19] movement disorders defined by sustained or transient involuntary muscle contractions. 20,21nvoluntary movements in BK GOF patients often initiate during childhood [17][18][19]22 and can co-occur with developmental delay, intellectual disability, and autism, 18,22 suggesting that BK GOF may perturb neural development.However, while knock-in murine models of BK GOF have recently been generated and exhibit motor defects, 23,24 it remains unclear whether GOF BK channels act during critical developmental periods, or in the fully developed nervous system, to disrupt movement.With its wealth of tools enabling spatiotemporal manipulation of gene expression, the fruit fly, Drosophila, represents an ideal model with which to examine this question.Stimulus-independent neural activity has been observed during development of the adult Drosophila nervous system, 25,26 and the Drosophila genome encodes a BK channel a-subunit termed SLOWPOKE (SLO) that is highly homologous to its human hSlo1 ortholog.][29][30] Flies heterozygous for the equivalent amino-acid change (E366G) in SLO (slo E366G/+ ) exhibit an array of motor phenotypes, including reduced total movement, reduced locomotor speed, and bouts of dyskinesia-like leg twitches, 31 providing robust phenotypic readouts of the impact of BK GOF on motor control.In this background, we developed a genetic strategy that allowed us to reversibly promote or inhibit expression of neuronal GOF BK channels.Combining this method with measurements of locomotor activity, limb kinematics, synaptic protein expression, and neurotransmitter release, we show that GOF BK channels act during a critical period during the final stages of neurodevelopment to disrupt limb control in adult flies, and we provide evidence that this occurs-at least in part-by BK GOF perturbing neuronal activity and synaptic maturation during this period.

Drosophila neurons
We sought to define the relative impact on movement of expressing GOF BK channels in developing versus mature neurons.To do so, we developed a method to reversibly tune neuronal levels of SLO BK channels through post-translational control.We based our approach on our studies of dyschronic (dysc), a scaffold protein that binds to SLO BK channels and promotes their expression.Brain-wide SLO expression is greatly reduced in flies homozygous for a dysc loss-of-function (LOF) allele (dysc s168 ), 32 and we recently showed that homozygosity for dysc s168 suppressed motor defects in slo E366G/+ flies by reducing the expression of GOF BK channels. 31Furthermore, restoring DYSC expression solely in post-mitotic neurons (nsyb-Gal4>UASdysc) reinstated locomotor defects in slo E366G/+ , dysc s168/s168 double-mutant flies, 31 showing that GOF BK channels act in post-mitotic neurons to disrupt movement.
In this background, we placed UAS-dysc under thermogenetic control using tub-Gal80 ts , 33 a globally expressed GAL4-inhibitor that blocks GAL4-driven transgene expression at low (18 C) but not high temperatures (29 C).This method-which has been extensively used to limit transgene expression to defined stages of the Drosophila life cycle [33][34][35][36] -should promote robust expression of DYSC, and therefore SLO BK channels, at the permissive temperature of 29 C and suppress expression at 18 C (Figures 1A and 1B; see STAR Methods).Advantageously, this approach also maintains the endogenous transcriptional and post-transcriptional regulation of slo expression 27,37 and an appropriate 50:50 ratio of wild-type to GOF BK channel subunit expression. 17or simplicity, we will henceforth use the terms robust BK channel expression on or off (''rBK ON/OFF'') to denote the temperature-dependent promotion or suppression of SLO BK channel expression via the above method.This will be performed in backgrounds heterozygous for the BK GOF allele (slo E366G/+ : rBK GOF ) or a genetically isogenized wild-type slo allele derived using the same homologous recombination process (slo loxP/+ : rBK WT ) 31 (see STAR Methods for full genotypic details).

GOF BK channels act during development to disrupt movement in Drosophila
To confirm that our approach yielded expected behavioral outcomes, we tested whether constitutively inhibiting neuronal rBK GOF expression throughout the Drosophila life cycle suppressed locomotor defects of slo E366G/+ flies. 31The number of infrared beam breaks made by flies housed in Drosophila activity monitor (DAM) 34 systems (Figure 1C) over 24 h-a measure of overall locomotor activity-is profoundly reduced in slo E366G/+ flies. 31As predicted, this phenotype was suppressed by inhibiting neuronal rBK GOF expression (Figures 1D and 1E).Conversely, constitutively promoting neuronal rBK GOF expression restored locomotor defects in slo E366G/+ flies, as shown by a significant reduction in beams breaks compared with relevant controls (Figures 1D and 1F).
We then examined whether we could define a key pathogenic time window in which neuronal BK GOF perturbs movement.We first tested whether inducing rBK GOF expression in the mature  S1.adult nervous system caused locomotor defects.Because synaptic growth during neurodevelopment extends into the first 1-2 days of adulthood in Drosophila, 38 we induced rBK GOF expression after 2 days of adulthood and maintained this for 3 days prior to locomotor analysis, allowing an extended period for GOF BK channels to accumulate and acutely induce motor deficits (Figure 1D; we define this condition as ''adult neurons'').The elevated daytime activity of dysc s168 homozygotes, which likely occurs due to reduced SLO expession, 32 was strongly suppressed by inducing rBK GOF and rBK WT expression in adultstage neurons (Figures S1A-S1F).However, adult-stage rBK GOF expression did not reduce total locomotor activity compared with controls (Figure 1G), indicating that while sufficient to impact some behaviors modulated by BK channels, this manipulation did not impair overall motor capacity.We therefore induced neuronal rBK GOF expression during either the pupal or the embryonic-to-larval stage of the fly life cycle (Figure 1D).We found that inducing neuronal rBK GOF (but not rBK WT ) expression during the pupal stage led to striking locomotor defects in resulting adult flies (Figure 1H), whereas induction during the embryonic-to-larval stages did not (Figure 1I).
As a complementary approach, we examined how discrete periods of BK GOF affected acute, stimulus-induced locomotor activity.We deployed a video-based system called Drosophila arousal tracking (DART) capable of quantifying the velocity of movement induced by a mechanosensory stimulus 39 (Figure 2A).Consistent with the above data, we found no difference in the stimulus-induced peak locomotor velocity of flies with rBK WT or rBK GOF expression induced in adult neurons (Figures 2B-2D).By contrast, inducing neuronal rBK GOF expression during the pupal stage strongly reduced locomotor velocity compared with controls (Figures 2B, 2E, and 2F).Thus, inducing GOF BK channel expression solely during the pupal stage is sufficient to disrupt both self-driven and stimulus-induced movement.
To test whether expression of GOF BK channels specifically during the pupal stage was necessary as well as sufficient to disrupt movement, we induced rBK GOF expression in all stages of the fly life cycle except the pupal stage.Using the DAM system, we observed a significant reduction in overall movement in flies with neural rBK GOF expression induced during the embryonic-to-larval and adult stages alone (Figures S2A and  S2B).However, this phenotype was far less severe compared with the converse experiment of inducing rBK GOF expression solely at the pupal stage, as quantified by Cohen's effect size d (embryonic-to-larval and adult-stage induction: d = À0.59;pupal induction: d = À2.62).Furthermore, DART recordings revealed that inducing rBK GOF expression in embryonic-to-larval and adult neurons increased the variability of stimulus-induced locomotor velocity but did not reduce mean velocity compared with controls (Figures 2G and 2H), in contrast to the effect of inducing neuronal rBK GOF expression during the pupal stage (Figures 2E and 2F).In concert, these data strongly indicate that GOF BK channels act during a pupal-specific developmental period to disrupt movement in adult Drosophila.

GOF BK channels act during the late stages of neurodevelopment to perturb movement
The transformation of the larval into the adult Drosophila nervous system during metamorphosis occurs via stereotyped, temporally organized phases of neuronal development, which include the pruning of existing larval dendrites and axons, the re-growth of dendritic/axonal arbors into their adult forms, and, finally, the formation and maturation of synaptic connections. 40,41We searched for clues as to which of these processes BK GOF might perturb.To do so, we induced neuronal rBK GOF expression during five 24-h windows coinciding with distinct neurodevelopmental transitions during the pupal stage (Figures 2I and 2J).Note that at 18 C, pupal development occurs at approximately 0.53 the rate of the standard growth temperature of 25 C, under which pupal development of males takes approximately 102 h. 42he 5 windows thus begin at the equivalent of 0, 36, 60, 72, and 84 h after pupal formation (APF) in standard conditions (equivalent hours, ehAPF).
We observed a clear time-dependent effect of neuronal rBK GOF expression during the pupal stage on subsequent adult fly movement (Figure 2J).Induction of neuronal rBK GOF expression during early-to-mid stages of the pupal stage either did not significantly reduce movement compared with controls lacking rBK GOF expression or reduced movement only to a small degree (Figure 2J).In contrast, induction of neuronal rBK GOF (I) Schematic illustrating induction of neuronal rBK GOF expression for 24-h periods during periods of the pupal stage (paradigms 2-6; paradigm 1 denotes continued suppression of rBK GOF expression).Experiments on adult male flies took place 5-7 days after eclosure.(J) Total beam breaks over 24 h recorded using the DAM system in the paradigms illustrated in (I).n = (left to right) 81, 18, 15, 28, 11, 12 flies.Statistical comparisons were made between paradigm 1 (negative control) and paradigms 2-6.Above graph: approximate timeline showing developmental processes overlapping with the period of GOF BK channel expression in each paradigm.S1. expression in neurons for a 24-h period 72 or 84 ehAPF profoundly reduced movement in resulting adult flies (Figure 2J).Inducing neuronal rBK WT expression for 24 h at 84 ehAPF, however, had no effect on overall movement (Figures S2C and S2D).We also confirmed the detrimental effect of inducing neuronal rBK GOF expression 84 ehAPF on movement using DART (Figures 2K and 2L).These data demonstrate that GOF BK channels act during a critical period within late neurodevelopment to impact movement in adult Drosophila.

Enhancing BK channel activity during the late pupal stage disrupts limb kinematics
We next sought to understand how BK GOF during neurodevelopment impacted movement at the level of individual limbs.Human carriers of the equivalent mutation to slo E366G/+ (hSlo1 D434G/+ ) present with bouts of involuntary dystonic and dyskinetic limb movements. 17slo E366G/+ flies exhibit similar bouts of leg twitches, consisting of rapid, repetitive movements of a single limb-a phenotype not observed in control flies. 31We asked whether inducing GOF BK channel expression during neurodevelopment also yielded dyskinesia-like phenotypes.Indeed, inducing neuronal rBK GOF expression at 84 ehAPF caused at least one bout of multiple leg twitches during a 5-min period in 66% of flies (n = 9), whereas inducing rBK GOF expression in adult neurons did not (n = 5; Figures S2E-S2H).Dyskinesia-like movements in the above backgrounds occurred while flies were at rest.Hence, this analysis did not reveal how alterations in limb kinematics contributed to the reductions in locomotor velocity observed in slo E366G/+ flies 31 and in flies with neuronal rBK GOF expression induced during the pupal stage (Figures 2E and 2F).To study this, we utilized feature-learning-based limb segmentation and tracking (FLLIT), a machine-learning image analysis package capable of deriving gait parameters from high-speed videography of fly locomotion 43 (Figure 3A; see STAR Methods, Wu et al., 43 and Mendes et al. 44 for descriptions of gait parameters).
Using FLLIT, we first examined limb movements in slo E366G/+ flies versus controls (Figures 3B and 3C).We uncovered changes in four kinematic parameters that likely contribute to the reduced movement and locomotor speed observed in slo E366G/+ flies. 31These are: a reduced percentage of time each limb spent moving (Figure 3D), reduced stride length (displacement; Figure 3E), reduced velocity of limb movement (Figure 3F), and an enhanced non-linearity of limb movement (calculated by normalizing the actual limb path to the idealized limb displacement) suggestive of uncoordinated limb movements (Figures 3B, 3C, and 3G).
We next investigated how inducing neuronal rBK GOF expression in adult neurons or during late neurodevelopment influenced these kinematic parameters.Consistent with results derived from DAM and DART systems, rBK GOF expression in adult neurons did not significantly alter limb kinematics compared with controls (Figures S3A-S3G).In contrast, 24-h induction of neuronal rBK GOF starting at 84 ehAPF significantly reduced the time each limb spent moving, reduced stride displacement and limb velocity, and enhanced the non-linearity of limb movement (Figures 3H-3N).We also noted a substantial increase in the variability of limb displacement and movement linearity in slo E366G/+ flies, quantified via the coefficient of variation (Figures S4A and  S4B).A small increase in the variability of limb movements was observed following induction of rBK GOF in adult neurons (Figures S4C-S4E), while a much greater increase was observed following induction of neuronal rBK GOF expression at 84 ehAPF (Figures S4F and S4G).
Collectively, these data demonstrate that inducing neuronal rBK GOF expression during the late pupal stage causes dyskinesia-like limb movements and recapitulates motor phenotypes caused by constitutive GOF BK expression, while expression in adult neurons does not.

BK GOF alters presynaptic protein expression during the late pupal stage
The critical neurodevelopmental window in which GOF BK channels act to perturb adult movement coincides with periods of neural arborization, synaptogenesis, and synaptic maturation within the pupal stage (Figure 3B), 40,41 suggesting that one or more of these processes may be disrupted.To test this, we first examined axonal and synaptic development in flies with constitutive GOF BK expression (slo E366G/+ ) compared with controls (slo loxP/+ ).We focused on two neural subtypes in the fly brainthe mushroom bodies (MBs) and large ventral lateral neurons (l-LN v s)-because SLO BK channels can be visualized in the MBs 32 and influence AP repolarization in the l-LN v s. 31,45 We found no difference in axonal growth of four MB compartments (the ab and a 0 b 0 lobes) between slo E366G/+ and control adult flies, nor were number or size of presynaptic l-LN v boutons innervating the fly optic lobe altered between slo E366G/+ and control flies (Figure S5).
These data suggest that GOF BK channels do not grossly disrupt neural arborization and synaptic growth in Drosophila.(D-G) FLLIT-derived gait parameters comparing slo loxP/+ and slo E366G/+ flies.Data points represent mean values from a single limb across 3-6 strides.n = 66 limbs across 11 flies (slo loxP/+ ) and 60 limbs across 10 flies (slo E366G/+ ).(H) Schematic illustrating paradigm to induce neuronal rBK WT or rBK GOF expression during late neurodevelopment.Experiments on adult male flies took place 5-7 days after eclosure.(I and J) Representative FLLIT-derived traces showing movement of each limb over 3-6 strides, relative to body center, for flies with neuronal rBK WT or rBK GOF expression induced during a 24 h period at the end of the pupal stage.(K-N) FLLIT-derived gait parameters for flies with neuronal rBK WT or rBK GOF expression induced during a 24 h period at the end of the pupal stage.n = 24 limbs across 4 flies (control, gray) and 42 limbs across 7 flies (BK GOF, orange).Error bars: 95% CI. *p < 0.05, ***p < 0.0005, ns p > 0.05, Mann-Whitney U test.See also Figures S3 and S4 and Tables S1 and S2.(legend continued on next page) We therefore focused our investigations on the final stages of synaptic maturation.][48] Hence, we attempted to first define cell types in which GOF BK channels act to perturb movement.We induced constitutive rBK WT and rBK GOF expression in four distinct cell types-chordotonal and multi-dendritic sensory neurons, all post-mitotic neurons, and post-mitotic neurons primarily outside of the ventral nerve cord (VNC)-using tsh-Gal80 as a suppressor of VNC transgene expression 49 (Figures 4A-4C, S6A, and S6B).Inducing rBK GOF in populations of sensory neurons did not impair movement, whereas induction of rBK GOF (but not rBK WT ) in post-mitotic neurons and post-mitotic neurons primarily outside of the VNC, did (Figures 4B and 4C).These data suggest that critical circuits in which GOF BK channels act to perturb movement in Drosophila likely reside in the developing adult brain.We thus tested for alterations in synaptic protein expression in pupal brains.Indeed, immunofluorescent imaging revealed an $35% reduction in synaptic expression of Bruchpilot (BRP), a scaffold protein that tethers synaptic vesicles in proximity to calcium channels within the active zone, 50 in late-stage slo E366G/+ pupal brains ($96 hAPF) (Figures 4D and 4E).This reduction appeared to be brain wide, occurring in eight neuropil regions that collectively comprise a substantial portion of the anterior brain surface (Figures S6C  and S6D).slo E366G/+ adults also exhibited a significant reduction in BRP localization compared with controls, albeit of lower magnitude (Figure 4E).In contrast, during the mid-pupal stage ($60 hAPF), synaptic BRP localization was similar between slo E366G/+ and control brains (Figure 4E).These data suggest that BK channel GOF leads to changes in presynaptic composition during a late-stage developmental period, which partially persist into adulthood.
We probed the mechanisms underlying reduced synaptic BRP localization in slo E366G/+ late-stage pupae and adults.Induction of neuronal rBK GOF expression solely during the pupal stage (<10-100 ehAPF) significantly reduced synaptic BRP localization in resulting adult brains (Figures 4F-4H), indicating that GOF BK channels act during neurodevelopment to influence adult synaptic BRP expression.Surprisingly, western blotting revealed that BRP protein levels were unchanged in slo E366G/+ compared with control late-stage pupal brains ($96 hAPF) (Figures S7A  and S7B).Additionally, the localization of discs large (DLG)-a post-synaptic pan-neuronal scaffold protein 51 -was unaltered in slo E366G/+ pupal brains at $96 hAPF (Figures S7C and  S7D).These data suggest that the reduction in synaptic BRP localization in slo E366G/+ late pupal brains is not driven by reduced BRP protein levels or synapse/neuron loss, but it may be indicative of decreased active zone number or an altered distribution of BRP within presynaptic domains 52 (see discussion).

BK channel GOF suppresses excitatory neurotransmission during development
Developing nervous systems are characterized by stimulus-independent neural activity that facilitates synaptic connectivity. 2,25,53,54Because BRP promotes neurotransmitter release at the Drosophila larval neuromuscular junction (NMJ), 50 reductions in presynaptic BRP levels in slo E366G/+ late-stage pupae might inhibit neurotransmitter release during this period, as could enhanced activity of BK channels localized to presynaptic domains. 12Hence, reducing neurotransmission during neurodevelopment represents a plausible mechanism by which BK channel GOF could cause long-lasting disruption of adult-stage motor control.
We therefore tested whether BK channel GOF suppressed neurotransmission during the late pupal stage using synapto-pHluorin (spH), a pH-sensitive GFP variant localized to the lumen of synaptic vesicles. 55Fusion of acidified vesicles with the presynaptic membrane results in local pH neutralization and an increase in spH fluorescence, providing an optical readout of neurotransmitter release (Figure 5A). 55Because excitatory transmission promotes neuronal Ca 2+ influxes during development, and acetylcholine is the predominant excitatory neurotransmitter in insects, we expressed spH in cholinergic neurons and imaged spH fluorescence in control and slo E366G/+ brains at $96 hAPF, using an ex vivo preparation to visualize stimulus-independent neurotransmission.As predicted, we observed a significant reduction in spH fluorescence across cholinergic synapses in slo E366G/+ late-stage pupal brains compared with controls (Figures 5B and 5C).Analysis of neuropil domains revealed that this reduction occurred in several brain regions that regulate movement and that are activated prior to or during locomotion, including the anterior optic tubercle, superior medial protocerebrum, and the MBs [56][57][58][59] (Figures 5D-5H and S7E-S7G).Thus, BK GOF widely inhibits excitatory neurotransmission during late neurodevelopment in Drosophila.

Reduced neural activity during development disrupts limb control in Drosophila
From the latter half of the pupal stage onward, patterned stimulus-independent neural activity (termed ''PSINA'') occurs widely through the developing Drosophila brain and influences synaptic maturation. 25 The above data suggested that the inhibition of PSINA by BK GOF might disrupt locomotion in adult flies.To examine the link between neural activity during development and movement, we expressed dOrkDC2-a constitutively active open rectifying channel that would be predicted to suppress PSINA by hyperpolarizing neurons 60 -in pupal post-mitotic neurons (Figure 6A).Approximately half of flies subjected to pupal pan-neuronal silencing (<10-100 ehAPF) failed to eclose from the pupal case, suggestive of extreme motor dysfunction.Using the DAM system, we indeed found that flies that survived this manipulation exhibited a significant reduction in movement compared with relevant controls (Figure 6B).
We next used the FLLIT system to test how limb kinematics were altered by pupal-stage neuronal silencing (Figures 6C-6H).Strikingly, three of four aspects of limb movements impaired in slo E366G/+ flies (the % of time each limb moved, displacement, and non-linearity of limb movement, but not limb velocity) were also perturbed by silencing neural activity during development (Figures 6E-6H).Furthermore, we noted an increase in the variability of limb displacement and movement linearity in flies subjected to pupal neuronal silencing (Figures S8A-S8C), as was observed in in slo E366G/+ flies and in flies with neuronal rBK GOF expression induced during the late pupal stage (Figure S4).Thus, reduced neuronal excitability during neurodevelopment, as is observed in slo E366G/+ pupae, is sufficient to impair movement in resulting adult flies by altering specific aspects of limb kinematics and movement stereotypy.

Elevating neural activity during development rescues motor defects in BK GOF flies
Finally, we sought to demonstrate a causal link between reduced neurotransmission during neurodevelopment and disrupted movement and limb kinematics in slo E366G/+ flies by testing whether increasing the excitability of pupal neurons could rescue movement defects in slo E366G/+ adults.We utilized TrpA1-a thermo-sensitive cation channel mildly activated at 27 C and strongly at 29 C 61 -to elevate the activity of pupal neurons in slo E366G/+ and control backgrounds.Initially, we strongly elevated activity of all cholinergic neurons in slo E366G/+ and control pupae, but this manipulation was largely lethal in the slo E366G/+ background, with very few flies eclosing.Because this suggested that BK GOF flies were particularly sensitive to the levels of neural activity during development, we induced a more subtle manipulation.Recent work has identified a small subpopulation of neurons defined by expression of the Trp-g cation channel that promote PSINA during development 26 (Figure S8D).We used a reporter of Trp-g expression that labels neurons in the gnathal ganglia, optic lobes, and superior protocerebral domains of the pupal brain 62,63 (Figures S8E and  S8F).We drove TrpA1 expression in Trp-g-neurons labeled by this reporter, and induced mild or strong activation of TrpA1 throughout the pupal stage (<10-100 ehAPF) in control and slo E366G/+ backgrounds (Figures 7A and 7B).In slo loxP/+ controls, we observed a negative effect of high temperature during the pupal stage on adult movement, with no specific impact of activation of Trp-g-neurons being apparent (Figure 7B).In contrast, in the slo E366G/+ background, both mild and strong elevation of Trpg-neuron activity significantly enhanced overall movement compared with slo E366G/+ flies expressing TrpA1 channels that were inactive during neurodevelopment, with strong activation of Trp-g-neurons during development inducing the most robust rescue (Figure 7B).
We next tested how strong Trp-g-neuron activation during the pupal stage affected limb kinematics and dyskinesia-like limb movements in slo E366G/+ flies.We found that three out of the four kinematic parameters perturbed in slo E366G/+ flies were partially rescued, with pupal Trp-g-neuron activation inducing an increase in the % of time each limb was moving, an increase in stride displacement, and an increase in the linearity of limb movement (Figures 7C-7F and 7H).Notably, these same kinematic parameters were disrupted by neuronal silencing during the pupal stage (Figures 6E, 6F, and 6H).In contrast, the limb velocity of slo E366G/+ flies-a kinematic parameter unaffected by neural silencing during the pupal stage (Figure 6G)-was unaltered by pupal Trp-g-neuron activation (Figure 7G).Nor did we observe a substantial reduction in the variability of limb displacement, the non-linearity of limb movement, or the number or duration of dyskinesia-like leg twitches in slo E366G/+ flies following activation of pupal Trp-g-neuron (Figures S8G-S8K).
These data reveal specific aspects of the multi-faceted motor phenotype of BK GOF flies that are sensitive to neuronal activity occurring during development of the adult Drosophila nervous system.

DISCUSSION
Neural activity occurring during development regulates the formation and patterned output of vertebrate motor circuits. 7,9,64,659][70] However,  S1. (legend continued on next page) holometabolous insects such as Drosophila undergo a second period of neurodevelopment prior to adulthood-the pupal stage-during which the larval nervous system undergoes metamorphosis to create an adult organization. 40,41Stimulus-independent neural activity also occurs in the pupal brain. 25,26Yet, whether such activity influences movement in adult flies is poorly understood, and critical periods during the development of the adult nervous system remain ill-defined.More broadly, whether disruption of ion channels can perturb critical periods of vertebrate or invertebrate motor development is largely unclear, despite the relevance of this question to earlyonset movement disorders in humans caused by ion channel mutations. 21,71ere, we study how motor output in Drosophila is disrupted by a particular class of ion channels-BK channels-harboring a mutation linked to two involuntary movement disorders, dystonia and dyskinesia. 17,31We identify a critical period in the late pupal stage during which neural development becomes sensitive to BK GOF.Furthermore, we uncover alterations in synaptic protein expression and neurotransmission during this developmental window and delineate causal relationships between specific aspects of adult fly limb movements and the levels of neural activity occurring during neurodevelopment.

Defining the critical period
Our core results derive from a method of promoting or inhibiting BK channel expression that involves spatiotemporal manipulation of the SLO-binding protein DYSC. 32Two caveats of this approach should be highlighted.First, our method necessitated that we perform behavioral experiments in a dysc mutant background.Because dysc mutants exhibit defects in circadian patterns of locomotion 32 and fly movement was measured by the DAM system over a 24-h period, alterations in circadian activity could yield confounds.We control for this using two video-based methods that directly measure movement capacity and limb kinematics: the DART and FLLIT systems. 39,43Data derived from these systems support a pathological impact of BK GOF during development (Figures 2 and 3).
Second, because the degradation rates of DYSC and SLO proteins are unknown, BK channels induced solely during the pupal stage could be partially retained into adulthood, making it difficult to rule out the possibility that BK GOF is required in both pupal and adult neurons to promote motor dysfunction.Nonetheless, observations from slo E366G/+ flies (wild type except for a heterozygous mutation in slo) support a neurodevelopmental impact of BK GOF.We observed a reduction in presynaptic BRP in slo E366G/+ neurons that emerged during, but not before, the critical period identified by behavioral analyses of flies subject to induced alterations in DYSC and SLO expression (Figure 4).Furthermore, enhancing neural activity during the pupal stage partially rescued motor defects in slo E366G/+ adult flies (Figure 7).Because this intervention involves rapid activation/inactivation of TrpA1 cation channels, 61 direct effects on adult-stage neuronal excitability can be excluded.These complementary datasets support our main conclusion: that BK GOF impairs limb control in Drosophila by perturbing neuronal development, in part by inhibiting neurotransmission in the developing brain.

BK channels and neurodevelopment
Prior work has provided tantalizing links between BK channel activity and neurodevelopment.Studies of developing vertebrate cell types, such as chick lumbar motoneurons, mouse cochlear inner hair cells, and rat neocortical pyramidal and Purkinje neurons, have revealed dynamic increases in BK channel expression during development that are gated by neural activity, 15,16,[72][73][74] potentially representing a negative feedback loop in which BK channels terminate spontaneous activity or switch neural firing from immature rhythmic to mature bursting patterns. 2In concert with their ability to influence short-term, 75,76 long-term, 13,77 and homeostatic forms of synaptic plasticity, 14 BK channels may thus impact critical periods by regulating both the duration of spontaneous neural excitability and the degree of plasticity occurring during these developmental windows.
A neurodevelopmental impact of BK channels is also consistent with studies of humans harboring BK channel GOF mutations.Involuntary movements in human hSlo1 D434G carriers initiate mainly before the age of 6 (in some cases less than 6 months after birth). 17Hence, key pathogenic processes that cause loss of limb control in hSlo1 D434G carriers occur during neurodevelopment.Furthermore, while hSlo1 D434G carriers do not present with overt neurodevelopmental phenotypes, hSlo1 mutations such as N999S, which enhance BK channel activity to a greater degree than D434G, are associated with developmental delay, intellectual disability, and autism spectrum disorder. 22,78,79nterestingly, a comprehensive study by Dong et al. identified locomotor defects in knock-in mice carrying the equivalent KCNMA1 D434G allele that were partially rescued by intraperitoneal injection of Paxilline, a BK channel blocker. 23While these results imply an acute role for BK GOF in causing locomotor defects in mice, our results suggest an alternative interpretation: that modulating AP frequency or neurotransmission via BK channel inhibition may counteract the effect of prior neurodevelopmental perturbations caused by BK GOF.Notably, Dong et al. identified alterations in the somatic and dendritic morphology of cerebellar Purkinje neurons in KCNMA1 D434G mice, 23 supporting a link between BK GOF and altered neurodevelopment in mammals.An important extension of our study will therefore be to develop genetic methods to induce GOF BK expression in the developing or developed nervous system in BK GOF mouse models 23,24 and define the resulting impact on motor phenotypes.
(B) Total beam breaks over 24 h recorded using the DAM system in the paradigms illustrated in (A).tub-Gal80 ts , nsyb-Gal4/+: paradigm 1, n = 16; paradigm 2, n = 20.tub-Gal80 ts , nsyb-Gal4>UAS-OrkDC2: paradigm 1, n = 15; paradigm 2, n = 6.(C and D) Representative FLLIT-derived traces showing movement of each limb over 3-6 strides, relative to body center, for control (tub-Gal80 ts , nsyb-Gal4/+) (C) and experimental (tub-Gal80 ts , nsyb-Gal4>UAS-OrkDC2) (D) flies subject to experimental paradigm 2. (E-H) FLLIT-derived gait parameters comparing control to experimental flies.Data points represent mean values from a single limb across 3-6 strides.n = 42 limbs across 7 flies for both genotypes.Error bars: 95% CI. *p < 0.05, ***p < 0.0005, ns p > 0.05, two-way ANOVA (B) or Mann-Whitney U test (E-H).See also Figure S8 and Tables S1 and S2.(legend continued on next page) Another goal will be to determine how excitatory neurotransmission and synaptic protein localization are perturbed by BK GOF.Super-resolution imaging has revealed that loss of BK channels enlarges BRP-labeled active zones at the larval Drosophila NMJ 80 and that altered compaction of active zones can change the immunofluorescence of their protein constituents when visualized through confocal microscopy. 52Alterations in active zone structure could explain why BRP immunofluorescence is reduced in pupal slo E366G/+ neurons despite BRP protein levels not decreasing (Figures 4 and S7).Alternatively, changes in the trafficking of BRP or a decrease in the number of active zones could drive reduced BRP immunofluorescence.Whether BK GOF influences BRP localization by reducing neurotransmission 26 or, instead, regulates BRP through activity-independent mechanisms to then reduce excitatory neurotransmission, also remains unclear.Defining in more detail how the structure of the active zone cytomatrix is modified by BK GOF will hence be a productive line of enquiry.
Our study suggests that BK GOF primarily acts via affecting synaptic maturation in brain circuits (Figure 4).However, due to the incomplete suppression of transgene expression in the VNC by tsh-Gal80 (Figures S6A and S6B), we cannot fully rule out a direct effect of BK GOF on VNC circuits that control movement and limb kinematics. 81BK GOF in brain circuits could also limit transmission of excitatory signals to VNC neurons during development, leading to non-cell-autonomous effects on their maturation.Indeed, in late-stage BK GOF pupae, we found that synaptic BRP expression was reduced in the gnathal ganglia (Figure S6D), a domain containing descending neurons that connect sensory and higher-order brain circuits with VNC neurons. 82urther studies are thus required to define key motor circuits perturbed by BK GOF.
We also note that not all aspects of altered movement and limb control in slo E366G/+ flies were modulated by enhancing neural excitability during development.4][85] Hence, BK GOF may cause specific impairments in limb control, such as the dyskinesia-like movements observed in slo E366G/+ flies, by disrupting processes, such as gene expression, metabolism, or protein degradation, during neurodevelopment.The generation of invertebrate and vertebrate animal models harboring BK channel GOF mutations 23,24,31 provides a means to test this hypothesis.

Critical windows in involuntary movement disorders
Recent work has revealed that neurological disorders lacking overt neurodevelopmental phenotypes may nonetheless have important developmental components.For example, human fetuses carrying pathogenic alleles linked to Huntington's disease (HD), a prototypical age-related disorder associated with involuntary movements, exhibit anomalies in cortical development. 86rthermore, mouse models of HD exhibit reduced excitatory cortical activity specifically in the first post-natal week, and pharmacologically elevating activity during this early period rescues sensorimotor defects in HD mice. 87Elegant work has similarly identified an early critical therapeutic window for isolated dystonia caused by a LOF mutation in the TorsinA AAA+ ATPase, showing that restoration of TorsinA expression in juvenile but not adult neurons rescued dystonia-like phenotypes in TorsinA LOF mice. 88Our work reveals the existence of an upper limit on developmental BK channel activity that, when exceeded, disrupts limb control in Drosophila, supporting the concept that disruption of critical periods of neurodevelopment contributes to the pathogenesis of movement disorders such as dystonia and dyskinesia. 89Our investigations also have relevance to neurodevelopmental disorders such as Angelman syndrome that are caused by LOF mutations in negative regulators of BK channel expression, and in which patients exhibit dystonic or dyskinetic movements. 90,91

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, James E.C. Jepson (j.jepson@ucl.ac.uk).

Materials availability
Drosophila strains can be requested from the lead contact, James E.C. Jepson (j.jepson@ucl.ac.uk).

Data and code availability
Raw microscopy and locomotor data will be supplied upon request by the lead contact, James E.C. Jepson (j.jepson@ucl.ac.uk).This paper does not report original code.Any additional information required to re-analyse the data reported in this paper is available from the lead contact upon request.Fluorescence intensity was quantified using ImageJ.Z-stacks of the whole brain were 3D-projected using a maximum intensity projection.From these whole-brain stacks, ROIs were drawn around the entire central brain (excluding optic lobes) or relevant neuropil regions with dominant contributions in full depth maximum intensity values (shown schematically in Figures 5, S6, and S7 as defined by Ito and colleagues 93 ).Mean fluorescence values were taken with background subtracted, using the same ROIs for the primary and counterstain channels, and values were normalised against mean fluorescence value of the counterstain.To control for differences in global staining, values were normalised to the mean values derived from controls dissected, stained and imaged on the same day.

Live imaging
Transgenic synapto-pHluorin (UAS-spH) was expressed in cholinergic neurons using the cha-Gal4 driver.Late-stage pupa ($96 hAPF) brains were dissected in external solution (in mM: 101 NaCl, 1 CaCl 2 , 4 MgCl 2 , 3 KCl, 5 glucose, 1.25 NaH 2 PO 4 , 20.7 NaHCO 3 , pH 7.2).Images were taken immediately (within 5 min) as above.ROIs were drawn around the whole central brain as above, or around neuropil structures identifiable within the image, backgrounds subtracted, and mean fluorescence values taken.Values were normalised to the mean values of controls taken during the same session.
Adult behavioural analyses DAM Drosophila Activity Monitor (DAM) analysis was performed as described previously. 31Briefly, three-to five-day old male flies were collected and loaded into glass behavioural tubes (Trikinetics inc., MA, USA) containing 4% sucrose and 2% agar and left for two full days to acclimatise at the relevant ambient temperature.On the third day the total number of beam breaks over 24 h was measured using Drosophila Activity Monitor (DAM, Trikinetics inc., MA, USA).Experiments were conducted in in 12L:12D at the indicated temperature.DART Five-to seven-day old male flies were loaded into behavioural tubes and maintained in conditions similarly to above, but with flies maintained for 24 h at the relevant temperature before mechanical stimuli were applied.Locomotor activity was recorded using the Drosophila ARousal Tracking (DART) system (BFKlabs) 39 as previously described. 31After 24 h acclimatisation, a single stimulus in the form of a mechanical vibration (a train of five 200 ms pulses separated by 800 ms intervals, set at the DART system's maximum intensity) was delivered at Zeitgeber Time 3 (ZT3), causing a robust locomotor response.Videos were taken using a USB webcam (Logitech) and speed (mm/s) was analysed by the DART system from absolute position, and binned in one min intervals.Flies were excluded from analysis if they exceeded an average of 1 mm / s speed in the five one-minute bins preceding the stimulus.The mean speed (mm/s) in the one-minute bin following the stimulus was analysed.FLLIT For FLLIT (Feature Learning-based LImb segmentation and Tracking) measurements, five-to seven-day old de-winged male flies were loaded into custom-made 2x2 cm arenas without anaesthesia.1000 fps videos consisting of flies walking in a straight line and taking at least three clear strides were taken using a Photron FASTCAM Mini UX50 High Speed Camera with a Sigma 105 mm Macro lens.Gait analysis was performed using the FLLIT system. 3 Where FLLIT generates data for parameters per stride, the first and last stride were excluded and a mean of the remaining values taken.Values for each leg were pooled, giving six values per fly.FLLIT parameters and definitions are described in detail in Table S2 (supplemental information).
Leg twitch analysis 5 min videos of flies in horizontally-placed behavioural tubes were taken with an android phone camera.Leg twitches were manually identified as per the criteria described previously 1 : briefly, ''leg twitches'' occurred only in a single limb, consisted of >1 repetitive movements of similar characteristics, did not involve grooming, and were not coincident with forward or backward locomotion of >1/2 body length.These leg twitches tended to occur in bouts; the number and mean duration of bouts per fly were quantified.

Additional notes on DYSC-based thermogenetic control of SLO BK channel expression
The dysc locus encodes a scaffolding protein containing three PDZ domains. 32DYSC proteins bind to SLO BK channels and promote their functional expression in fly neurons in a post-translational manner, a process that likely requires the third C-terminal PDZ binding domain of DYSC. 32The expression of DYSC isoforms containing this third PDZ domain can be blocked via homozygosity for a P-element inserted between exons encoding the 2 nd and 3 rd PDZ domains (dysc s168 ), 32 the LOF allele we use in this study.We note that, in dysc s168/s168 homozygotes, shorter DYSC isoforms containing PDZ domains 1 and 2 are still transcribed, but SLO BK channel expression is nonetheless substantially reduced. 32Other LOF alleles of dysc are available -for example, the null allele dysc c03838 . 32However, homozygotes for dysc null alleles exhibit reduced viability (personal observations) and reduced peak movement ability. 32By contrast, the overall activity in dysc s168 homozygotes -as quantified by the number of infrared beam breaks over 24 h measured by the Drosophila Activity Monitor (DAM) system 94 -is slightly higher than isogenic control flies, 31 illustrating that dysc s168 homozygotes (unlike slo E366G/+ BK channel GOF flies 31 ) do not exhibit severe motor defects.Hence, use of dysc s168/s168 homozygote flies as a genetic background suppressive for SLO BK channel expression enabled us to maintain the slo E366G/+ allele in a background with robust overall movement.This in turn allowed us to readily measure decreases in total movement caused by the re-expression of DYSC (and therefore SLO GOF BK channels harbouring the E366G mutation) during the key developmental window in which GOF BK channels act to perturb movement and limb kinematics.

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analyses were performed using Graphpad Prism.Data sets were tested for normal distributions using the Shapiro-Wilk test.Normally distributed data sets were tested for statistical differences using unpaired t-tests with Welch's correction for non-identical variance or one-way ANOVA with Dunnett post-hoc test, or two-way ANOVA.Non-normally distributed data sets were tested using Mann-Whitney U-test or Kruskal-Wallis test with Dunn's post-hoc test.Where appropriate, a Dunn-Sida ´k correction for multiple comparisons was performed.Binary data were compared using chi-squared tests.Cohen's effect size was calculated using the following formula: Cohen's effect size d = (M 2 -M 1 )/SD pooled , where M 2 and M 1 are means of experimental vs control datasets, and SD pooled = O((SD 1 2 + SD 2 2 )/2), where SD 1 + SD 2 are the standard deviation of the experimental and control datasets.The coefficient of variation (CV) was calculated using the formula CV = SD/M, where SD is standard deviation of the data and M is the mean of the data.

Figure 1 .
Figure 1.Expression of GOF BK channels during neurodevelopment reduces spontaneous locomotion (A and B) Schematic illustrating a genetic method enabling temperature-dependent control of BK channel expression.(C) Schematic illustrating the DAM system and representative data.Flies are loaded individually into behavioral tubes.The DAM system records the number of times an individual fly breaks an infrared beam over 24 h in a 12-h light:12-h dark cycle.(D) Schematic illustrating developmental stages during which neuronal rBK WT or rBK GOF expression is induced.Letters refer to data shown in subsequent (E)-(I).Experiments on adult male flies took place 5-7 days after eclosure.(E-I) Total number of beam breaks in a 24-h period, recorded using the DAM system, in the paradigms illustrated in (D).Experimental genotype is denoted in orange, control genotypes in gray.Developmental stages during which rBK expression is induced are noted above each dataset.Error bars: 95% confidence interval (CI).*p < 0.05, ***p < 0.0005, ns p > 0.05, Kruskal-Wallis test with Dunn's post hoc test (E, F, H, and I) or one-way ANOVA with Tukey's multiple comparisons test (G).n values are: (E) n = (left to right) 46, 32, 15, 69 flies; (F) n = 35, 11, 17, 32 flies; (G) n = 24, 33, 36, 27 flies; (H) n = 13, 40, 10, 10 flies; and (I) n = 10, 28, 10, 22 flies.See also Figure S1 and TableS1.

Figure 2 .
Figure 2. Expression of GOF BK channels during a narrow developmental window impairs stimulus-induced and spontaneous movement (A) Schematic illustrating a stimulus-dependent locomotor assay: the Drosophila arousal tracking (DART) system.(B) Schematic illustrating developmental stages during which neuronal rBK WT or rBK GOF expression is induced.Letters refer to data shown in (C)-(H).Experiments on adult male flies took place 5-7 days after eclosure.(C, E, and G) Traces showing average speed over time in 1 min bins (mm/s) for control (n = 7) and slo E366G/+ (n = 11) flies in the paradigm shown in (B).Mechanical stimulus was delivered at 10 min.(D, F, and H) Quantification of speed following mechanical stimulus.Each data point represents average speed (mm/s) in the 1 min period following stimulation for each fly.Data are normalized to the control mean.n = 7, 11 flies.(I)Schematic illustrating induction of neuronal rBK GOF expression for 24-h periods during periods of the pupal stage (paradigms 2-6; paradigm 1 denotes continued suppression of rBK GOF expression).Experiments on adult male flies took place 5-7 days after eclosure.(J) Total beam breaks over 24 h recorded using the DAM system in the paradigms illustrated in (I).n = (left to right) 81, 18, 15, 28, 11, 12 flies.Statistical comparisons were made between paradigm 1 (negative control) and paradigms 2-6.Above graph: approximate timeline showing developmental processes overlapping with the period of GOF BK channel expression in each paradigm.(K) Traces showing average speed over time in 1 min bins (mm/s) for adult flies with rBK WT (n = 12) or rBKGOF  (n = 17) induced during late neurodevelopment.Mechanical stimulus is delivered at 10 min.(L) Quantification of speed following mechanical stimulus.Each data point represents average speed (mm/s) in 1 min bin following stimulation for one fly.Data are normalized to the control mean.n = 12, 17.Error bars: 95% CI. n values: (C and D): n = 7 slo loxP/+ , 11 slo E366G/+ ; (E and F): n = 19, 21; (G and H): n = 10, 7. **p < 0.005, ***p < 0.0005, ns p > 0.05, t test with Welch's correction (D), Mann-Whitney U test (F, H, and L), or Kruskal-Wallis test with Dunn's post hoc test.See also Figure S2 and TableS1.

Figure 3 .
Figure 3. BK channel GOF during late neurodevelopment disrupts limb kinematics (A) Schematic illustrating FLLIT methodology.Schematic on right shows overlaid movement of each limb over multiple strides, relative to body center.(B and C) Representative FLLIT-derived traces showing movement of each limb over 3-6 strides, relative to body center, for control (slo loxP/+ ) (B) and BK GOF (slo E366G/+ ) (C) adult male flies.(D-G)FLLIT-derived gait parameters comparing slo loxP/+ and slo E366G/+ flies.Data points represent mean values from a single limb across 3-6 strides.n = 66 limbs across 11 flies (slo loxP/+ ) and 60 limbs across 10 flies (slo E366G/+ ).(H) Schematic illustrating paradigm to induce neuronal rBK WT or rBK GOF expression during late neurodevelopment.Experiments on adult male flies took place 5-7 days after eclosure.(I and J) Representative FLLIT-derived traces showing movement of each limb over 3-6 strides, relative to body center, for flies with neuronal rBK WT or rBK GOF expression induced during a 24 h period at the end of the pupal stage.(K-N) FLLIT-derived gait parameters for flies with neuronal rBK WT or rBK GOF expression induced during a 24 h period at the end of the pupal stage.n = 24 limbs across 4 flies (control, gray) and 42 limbs across 7 flies (BK GOF, orange).Error bars: 95% CI. *p < 0.05, ***p < 0.0005, ns p > 0.05, Mann-Whitney U test.See also FiguresS3 and S4and TablesS1 and S2.

Figure 5 .
Figure 5. BK channel GOF reduces excitatory neurotransmission during late neurodevelopment (A) Schematic illustrating mode of action of synaptopHluorin (spH).Enhanced fluorescence is indicative of increased synaptic vesicle release.(B) Representative images of spH expressed in excitatory cholinergic neurons cha-Gal4 in control (cha>spH, slo loxP/+ ) and BK channel GOF (cha>spH, slo E366G/+ ) late-stage pupal brains.Scale bar: 50 mm.(C) Quantification of spH fluorescence in slo loxP/+ (n = 14) and slo E366G/+ (n = 13 brains) central brain regions of late-stage pupae.Data are normalized to the control mean.(D-H) Quantification of spH across neuropil domains in slo loxP/+ and slo E366G/+ ex vivo late-stage brains.Data are normalized to slo loxP/+ means within each neuropil domain.Location of each domain in the late pupal brain is shown below each dataset.Each data point represents data from a neuropil domain from an individual hemisphere.Abbreviations: AOTU, anterior optic tubercle; SMP, superior medial protocerebrum; AVLP, anterior ventrolateral protocerebrum; ALs, antennal lobes; hMBLs, horizonal mushroom body lobes.n = 26-30 per neuropil domain.Error bars: 95% CI. *p < 0.05, **p < 0.005, t test with Welch's correction or Mann-Whitney U test for parametric and non-parametric datasets, respectively.p values in (D)-(H) were subject to a Dunn-Sida ´k correction for multiple comparisons.See also Figure S7 and TableS1.

Figure 6 .
Figure 6.Suppressing neuronal excitability during late development disrupts locomotor activity and limb kinematics (A) Schematics illustrating the temporal pan-neuronal expression of OrkDC2.OrkDC2 expression during the pupal stage was terminated shortly before eclosure (paradigm 2).Experiments on adult male flies took place 5-7 days after eclosure.

TABLE d
92perimental models were fruit flies of the species Drosophila melanogaster.Flies were maintained on standard fly food under 12 h: 12 h light-dark cycles (12L: 12D).Unless otherwise indicated flies were maintained at a constant 25 C.For temperature manipulations they were maintained at 18 C, 27 C or 29-30 C at the time stages indicated.Experiments were conducted at the developmental stage indicated.Experiments were conducted on male flies.To control for genetic background all lines used were backcrossed into the isogenic w 1118 (iso31) background for at least 5 generations.The genotypes used, their function, and the experiments each was utilised for are described in TableS1(supplemental information).Brains were dissected in phosphate-buffered saline (PBS) (Sigma Aldrich) at the time point indicated (3-7 day old adult, late pupal stage [$ 96hAPF] or mid-pupal stage [$ 60hAPf]) and immuno-stained as described previously.92Briefly,brains were fixed by 20 min room temperature incubation in 4% paraformaldehyde (MP biomedicals), and blocked for one hour in 5% normal goat serum in PBS + 0.3% Triton-X (Sigma-Aldrich) (PBT).Brains were incubated overnight in primary antibody at 4 C, washed three times in PBT, and incubated overnight in secondary antibody at 4 C.Primary antibodies used were: mouse anti-BRP (nc82) at 1:500, mouse anti-discs large at 1:1000, mouse anti-PDF (PDF C7) at 1:100, mouse anti-FASII (1D4) at 1:200 (all from Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA; AB Registry ID: AB_528484).Secondary antibodies were goat anti-mouse AlexaFluor 488, 568, and 647 at 1:1000 and goat anti-rat AlexaFluor 647 at 1:1000 (ThermoFisher Scientific).Counterstains were DAPI at 1:1000 (ThermoFisher Scientific) and rat anti-Cadherin (DN-EX#8) at 1:500 (Developmental Studies Hybridoma Bank).Brains were mounted and imaged in SlowFade Gold anti-fade mountant (Thermofisher).Images were taken with a Zeiss LSM 710 confocal microscope with an EC 'Plan-Neofluar' 20x/0.50M27 air objective, taking z-stacks through the entire brain with step sizes of 1-2 mm.