Abstract
Building arborisations of the right size and shape is fundamental for neural network function. Live imaging studies in vertebrate brains strongly suggest that nascent synapses are critical for branch growth during the development of axonal and dendritic arborisations. The molecular mechanisms underlying such ‘synaptotropic’ events are largely unknown.
Here we present a novel system in Drosophila for studying the development of complex axonal arborisations live, in vivo during metamorphosis. In these growing axonal arborisations we see a relationship between the punctate localisations of presynaptic components and branch dynamics that is very similar to synaptotropic growth described in fish and frogs. These presynaptic components however do not appear to represent functional presynaptic release sites and are not paired with clusters of neurotransmitter receptors. Pharmacological and genetic knockdowns of evoked and spontaneous neurotransmission do not impact the outgrowth of these neurons. Instead, we find that axonal branch growth is regulated by the dynamic focal localisations of synaptic adhesion proteins Neurexin and Neuroligin. These adhesion complexes provide selective stability for filopodia by a ‘stick and grow’-based mechanism wholly independent of synaptic activity.
Introduction
Neurons are the most structurally diverse and complex cell type we know of (Bullock and Horridge, 1965). Their tree-like arborisations are critical for collecting, integrating and disseminating information between different synaptic partners. During development, arborisations grow dynamically and this morphogenesis sets limits on their final location, possible synaptic partners and core electrophysiological properties (Chagnac-Amatai et al., 1990; Roberts et al., 2014). Although there are recognisable cell-type specific shapes, each individual neuron has its own unique pattern of branching and connectivity. The ‘generative rules’ for constructing complex arborisations are believed to be encoded by genetic algorithms that play out in different developmental contexts to produce distinct morphological types (Cuntz at al., 2010; Teeter & Stevens., 2011; Chen & Haas, 2011; Hassan & Heisinger, 2015). Which molecules and mechanisms underlie this remains a major unanswered question within neuroscience.
From observations on the earliest phases of motoneuron dendrite growth in the spinal cord Vaughn and colleagues forwarded the synaptotropic hypothesis (Vaughn et al., 1974 &. The synaptotropic hypothesis posits that the stability of growing axonal and dendritic branches is controlled by the selective stabilisation of processes by nascent synapses, encouraging growth into territories rich in potential synaptic partners. Berry and colleagues, working on Purkinje cells, forwarded the same idea, calling it the synaptogenic filopodial theory (Berry & Bradley, 1976). Since then, live imaging in vertebrate brains has revealed dynamic axonal and dendritic growth (Kaethner & Stuermer, 1992; Wu et al., 1999; Jontes et al., 2000; Hossain et al., 2012) where the arrival and localisation of synaptic machineries is correlated with branch stabilisation (Alsina et al., 2001; Niell et al., 2004; Meyer & Smith, 2006; Ruthazer et al., 2006). Data on Xenopus tectal neuron dendritic growth revealed a role for Neurexin (Nrx) and Neuroligin (Nlg) in branch dynamics, where Nrx-Nlg interactions are believed to direct the genesis and maturation of opposing hemisynapses, after which neurotransmission stabilises branches (Chen et al., 2010). The idea that synaptic transmission stabilises nascent contacts during elaboration is supported by a number of studies (Rajan et al 1999; Sin et al., 2002; Ruthazer et al., 2003; Haas et al., 2006; Ruthazer et al., 2006). In contrast, other work has suggested that activity has little impact on large-scale features of tree growth but plays a more nuanced role in refining structural connectivity during activity dependent plasticity (Verhage et al., 2000; Varoquoaeux et al., 2002; Hua et al., 2005; Fredj et al., 2010).
Two key questions arise from this work: firstly, are growing branches stabilised by local ‘synaptogenic’ events? Secondly, are such nascent synapses being ‘use-tested’ by conventional neurotransmission? Here we describe a new system that we have pioneered in Drosophila to explore these questions. Unlike in the fly embryo where arborisations are very small and built very rapidly (<4 hours), our system takes advantage of the large axonal arborisations of the pleural muscle motoneurons (PM-Mns) that are built over an extended period, during metamorphosis. Also, unlike the growth of larval motoneuron terminals, which grow incrementally to scale with the changes in muscle size during larval life, the PM-Mn axonal arborisations grow exuberantly, by trial-and-error, very much like complex neurons found in vertebrate central nervous systems.
During development we see a consistent relationship between the distribution of presynaptic machineries and branch dynamics similar to that found in the retinal ganglion cell axons in fish and frogs. Importantly, we find that branch growth is driven by dynamic complexes in synaptic partners that we term neuritic adhesion complexes (NACs). These NACs contain Nlg1 (postsynaptically), along with Nrx, Syd1 and Liprin-α (presynaptically), and act locally to stabilise filopodia by a ‘stick and grow’ mechanism without the need of synaptic vesicle release machinery or functional synapses.
Results
Establishing a new model for exploring complex arborisation growth
In a search for a system to study complex arbor growth live, in vivo we identified the axonal arborisations of the motoneurons that innervate the abdominal pleural muscles of the adult fly. We refer to these as the pleural muscle motoneurons (PM-Mns), the axonal arborisations of which are large, complex and easily accessible throughout metamorphosis. Importantly, the PM-Mn system allows one to genetically manipulate either synaptic partner whilst independently imaging the other.
Each adult abdominal hemisegment (from A1-A7) contains a pair of motoneurons that exit the nerve onto the lateral body wall. In the adult, each motoneuron forms an arborisation that innervates between 15 and 18 parallel muscle fibres that span dorso-ventrally, from the tergites to sternites (Figures 1A&B). In contrast to the rigid, target-specificity found in the larval neuromuscular system, the innervation of the pleural muscles shows variation between segments and between animals. Although no one-to-one neuron/muscle targeting is found, arborisations achieve a consistent spatial organisation with regularly sized, non-overlapping projection fields.
To gain insight into the global development of the PM-Mn axon terminals, we imaged hemi-segment A3 from 24h to 84h after puparium formation (APF) (Figure 1C). By 24h APF the majority of larval muscles and neurons that innervate them are removed by programmed cell death and phagocytosis (Currie & Bate, 1991). The dorsally projecting peripheral nerve maintains four contacts with the epidermis during pupariation and allows the continued innervation of the persistent larval muscles. At around 24h APF axonal regrowth commences with the emergence of filopodia-rich growth cones from the nerve. By 34h APF primary branches are established and festooned with filopodia. The two PM-Mns within each hemisegment segregate into anterior and posterior domains and increase in size and complexity until approximately 72h APF. Following this, there is a period of maturation during which varicosities form along all but the most proximal branches. By 84h APF the morphology of the arborisations is indistinguishable from that observed at eclosion.
Live imaging at 2-minute intervals revealed that branch growth is highly dynamic, involving large numbers of filopodia which continually explore the local environment (Figure 1D). This exploration takes place almost exclusively within the plane of the developing muscles. Throughout this article, we define and refer to filopodia and branches as follows. According to anatomical and molecular criteria, filopodia are considered to be thin (<0.3μm), motile protrusions with cytoskeletons comprised of parallel F-actin filaments (Mattila & Lappalainen, 2008). In contrast, branches are more voluminous, of higher calibre and less dynamic, with cores containing parallel microtubules and noticeably less F-actin. Using Lifeact, a fluorescently tagged cytoskeletal probe that reveals F-actin (Riedl et al., 2008), we see an enrichment in thin ‘filopodia-like’ structures (Figure 1E). In contrast, the plus-end microtubule binding protein CLIP-170 (Stramer et al., 2010), that reveals microtubules, is found in what we refer to as branches but is mostly excluded from filopodia. Growing neurites are dynamic and constantly changing, but every effort has been made to be consistent with our classification.
Longer imaging intervals revealed the more substantial structural changes to arborisations during early phases of growth (Figure 1F). This showed us that the majority of filopodia are transient since large numbers are generated and lost between frames. Despite this, a small number of filopodia persist, pioneering individual branches (white arrowheads). In addition to construction, branches also collapse back into single filopodia (yellow arrowhead), highlighting the instability and continuous remodelling of the arborisations at this early stage (see movie 1).
The distribution of presynaptic components during axonal branch growth
To explore the growth of PM-Mn axonal arborisations and their muscle targets we imaged both the neuron and muscle progenitors (myoblasts) during the earliest phases of outgrowth. At 35h APF neurons and myoblasts are directly apposed to one another (Figure 2A and movie 2). Growing axons are enveloped by clusters of myoblasts, while the more distal growth cones and filopodia extend over sheets of immature myotubes.
In light of this very close association between the synaptic partners, we asked whether presynaptic components are present in the distal branches at this early stage, since previous work in vertebrates has forwarded that nascent synapses play a key role in branch stabilisation. Using an RFP tagged version of Bruchpilot (BRP), a homolog of ELKS/CAST, expressed with OK371-GAL4 (Mahr & Aberle., 2006), we see distinct, punctate distributions in axon branches, with accumulations at branch nodes and at the base of filopodia (Figure 2B). An analysis of BRP::RFP puncta in axon terminals of 5 animals staged from 30h to 35h APF revealed that 85.1 ± 6.4% (SD) of total BRP puncta localised at branch points and the base of filopodia, and 81.6 ± 4.6% of branch points/bases had a punctum (Figure 2C).
To look at the dynamics of BRP::RFP and branching we imaged PM-Mn arborisations at 2-minute intervals. This showed that BRP::RFP puncta are rapidly recruited to the tips of growing branches where they mark the sites of new filopodia growth (Figure 2D). Successive rounds of filopodia extension and stabilisation in this manner produce branches studded with BRP::RFP puncta. Filopodia which are generated from these puncta can give rise to new branches (Figure 2E).
The dynamics of BRP::RFP puncta in these time-lapse sequences speaks to a relationship between arbor growth and the distribution of presynaptic proteins (Figure 2F). To quantify this we analysed 83 filopodia generation events from 3 time-lapse movies of arborisations staged between 30h and 33h APF. Our analysis showed that 36.0 ± 12.6% of filopodia emerged from existing BRP puncta, 48.1 ± 10.7% recruited a punctum within 20 minutes of their formation and 16.0 ± 11.9% failed to recruit an obvious punctum at all (Figure 2G). The frequency of filopodia emerging from or recruiting a BRP::RFP punctum 71.4 ± 11.0% was significantly greater than a conservative expected frequency of 50% (X2 = 36.4, p < 0.0001). In addition, we found that 100% of filopodia that failed to recruit puncta to their bases were eliminated within 20 minutes of their emergence (n = 24) (Figure 2H). In contrast, although 54.2% of filopodia that originate from, or recruited BRP::RFP puncta were also lost within 20 minutes, 33.3% survived between 20 and 60 minutes and a further 12.5% persisted for over an hour (n = 24). Thus, the lifetimes of filopodia associated with puncta at their bases were significantly longer than those without (Mann-two-tailed Whitney U = 73, p < 0.0001) (See movie 3).
To verify the accuracy of GAL4 driven BRP::RFP, we imaged endogenous BRP protein tagged with GFP (Figure 2I) as immunohistochemistry is particularly difficult on fragile, early pupal body walls. The BRP::GFP protein trap revealed a punctate distribution at branch points and base of filopodia, as we had seen with the BRP::RFP reporter. Other GFP tagged protein trap lines that label synaptic vesicle (SV) associated proteins Syt1 and VGlut showed less punctate localisations, yet were still clearly concentrated in axon branches at the base of filopodia (Fig 2I). Interestingly we never saw these proteins within filopodia.
We wondered whether this node localisation of pre-synaptic machinery in the growing branches was specific to PM-Mns or is more widespread in the fly nervous system. To test this we imaged the output terminals of Eve (Even-skipped) positive interneurons in the thoracic neuromeres during metamorphosis (24h APF) (Roy et al., 2007). In these we found very similar distributions to those of the PM-Mns, with 79.5 ± 3.7% of puncta found at branch points or the base of filopodia (n = 8 output fields from 4 individuals) (Figures 2J&K).
PM-Mns elaborate their axonal arborisations without synaptic activity
The localisation and dynamics of presynaptic machineries described above mirrors that seen in Xenopus and zebrafish tecta and suggested to us a possible role for synapses and synaptic activity in branch growth during PM-Mn elaboration (Alsina et al., 2001; Niell et al., 2004; Meyer & Smith, 2006; Ruthazer et al., 2006).
To explore the role of neural activity in arborisation growth we mapped the electrical development of the PM-Mns using the genetically encoded calcium indicator GCaMP6m as a proxy (Movies 4-6). Calcium activity, recorded as changes in fluorescence, is shown as heat-registered kymographs at different stages of development (Figure 3A). The absence of any overt changes in fluorescence at 32h APF indicates that at this stage the motoneurons are electrically inactive. It was not until 42h APF that we saw the first calcium events indicative of membrane depolarisations. These events were isolated and infrequent. The frequency of calcium events increased sharply and by 48h APF activity was characterised by episodes of transients in quick succession lasting around 8 minutes, interspersed with longer periods of quiescence. Approaching the final stages of arbor growth, at 74h APF activity continued to be defined by similar periods of activity and quiescence, but at these stages bouts contained far greater numbers of events.
To address directly whether arbor growth can take place independently of activity-evoked neurotransmission we silenced neurons by intravital injection of the sodium channel blocker tetrodotoxin (0.05mM TTX). Of the 8 animals injected between 24h and 32h APF, 6 showed no detectable calcium activity at 78h APF (Figure 3B) and 2 showed very weak transients restricted to small parts of branches. In contrast, buffer injected controls gave expected levels of presynaptic activity (n = 5). At 79h APF we found the arborisations of animals injected with TTX to be morphologically indistinguishable from those injected with buffer (Figures 3C&D).
In addition to evoked neurotransmission, spontaneous neurotransmitter release, which is responsible for miniature postsynaptic potentials (mEPSPs), has been shown to play a key role in the growth and development of neurons (Choi et al., 2014; Andreae & Burrone, 2015). To determine if spontaneous neurotransmission is important for the growth of PM-Mn axonal arborisations we generated PM-Mn clones homozygous mutant for the vesicular glutamate transporter (VGlut) using the null allele Df(2L)VGlut2. This allele has been previously shown to completely block both evoked and spontaneous glutamatergic neurotransmission (Daniels et al., 2006). Antibody staining confirmed that Df(2L)VGlut2 MARCM clones have a complete loss of VGlut protein in motoneurons (Figure 3E). To assess the effect of removing VGlut on arbor growth we performed morphometric analysis on clones of the anterior-most motoneurons of segment A3 (A3-A) in females staged between 80h and 90h APF (Figures 3F&G). No significant difference was found between the area of coverage of VGlut null A3-A arborisations and controls (Figure 3H), although VGlut nulls were marginally greater in total arborisation length (Figure 3I).
The timeline of neural activity and the VGlut null data strongly suggests that synaptic transmission does not play a role during the ‘elaborative phase’ of growth of the pleural neuromuscular system. To determine when synaptic transmission is ‘physiologically’ possible we artificially stimulated neurons using the warmth-gated ion channel TRPA1 (Hamada et al., 2008), and measured postsynaptic calcium responses in the pleural muscles with GCaMP6m. Before 49h APF no calcium events were recorded in the muscles (n = 2) (Figure 3J). Between 50h and 59h APF a few large calcium events were recorded in the muscles but in each case these occurred either during the final seconds of stimulation or just after ramping down the temperature (n = 2). In contrast, when stimulating at stages between 68h and 72h APF we found robust, rapid and sustained postsynaptic calcium activity (Figures 3J&K). Between 68-72h APF calcium events occurred at a significantly greater rate at the permissive temperature (30°C) than at the restrictive temperature (22°C) (6.15 ± 5.60 minute-1 vs 0.93 ± 0.81 minute-1, Mann-Whitney U = 6.0, n = 7, p = 0.02, two-tailed). These data indicate that synaptic transmission does not take place within this system before 60h APF.
The lack of impact on growth from removing synaptic transmission made us look more closely at the organization of presynaptic machineries at different times during development. To do this we measured the distribution of BRP::RFP puncta in the same arborisation at 12 hour intervals from 34 to 58h APF (Figure 3L). As described previously, at 34h APF BRP::RFP puncta are found almost exclusively at branch points and at the bases of filopodia (white arrowheads). By 48h APF, in addition to the puncta at branch points, many puncta are also found along the lengths of branches, between branch nodes (yellow arrowheads). By 58h APF all but the most proximal branches are lined with large numbers of BRP::RFP puncta. Finally, an analysis of the distribution of puncta at stages 72-77h APF found that just 12.6 ± 5.8%, n = 5 arborisations of individuals) of puncta are at branch points, the rest being distributed along branch lengths (Figure 3M). The majority, 89.4 ± 6.7%), of branch points were still found to have puncta.
In addition to distribution, we used the BRP::GFP protein trap line to measure changes to BRP puncta size (Figures 3N&O). At early stages, BRP::GFP puncta diameters were found to be far more heterogenous, shown by the difference in standard deviation between the groups, found to be significant by an F-test of equality of variances (F143,176 = 4.092, p < 0.0001). We also found that BRP::GFP puncta became significantly smaller over the course of development (Figure 3O).
These smaller and more homogenously sized BRP puncta in later development highlighted that the puncta we see in early PM-Mn arborisations may not actually be synapses. To explore this idea we asked when glutamate receptors appear in the muscles. No detectable GluRIIE was evident at 48h APF (Figure 3P) yet by 85h APF clear clusters of GluRIIE were found apposed to the branches (Figure 3Q). Thus, the changes in the organisation of synaptic machineries over the course of development suggest that the early accumulations of presynaptic components do not represent differentiated synapses.
A role for Neuroligin1-Neurexin in PM-Mn arborisation growth
Neurexin-Neuroligin signalling is capable of driving synapse formation in many contexts and in the Xenopus tectum regulates arbor growth in an activity dependent manner (Haas et al, 2010). Though our data ruled out activity playing a key role in PM-Mn axonal arbor growth, we next sought to test whether these synaptic adhesion proteins are important.
We generated homozygous mutants using the null alleles, Nlg1e×2.3, Nlg11960 (Banovic et al., 2010) and Nrx241 (Li et al., 2007) in combination with chromosomal deficiencies. These combinations are viable into adulthood. The arborisations of both Nlg1 and Nrx mutants showed comparable defects with large reductions in coverage relative to wild-type controls (Figures 4A&B).
Morphometric analysis showed areas of coverage of Nlg1 and Nrx null arborisations were significantly lower than wild-type controls (Figure 4C), as were total arbor lengths (Figure 4D). Interestingly however, there was no difference in the total branch numbers (Figure 4E). Nrx and Nlg1 null arborisations were not significantly different from each other in any of these measurements. To determine if arbor complexity was different in the nulls we used the Strahler method of branch ordering (Figure 4F). Firstly, the groups showed no differences in their number of branch orders. Secondly, the total number of lowest order (terminal) branches was not significantly different between conditions; a trend which continued for subsequent orders, with each group showing remarkably similar numbers of branches at every level.
The morphology of late stage Nlg1 and Nrx nulls points toward a requirement for these proteins but does not tell us when they are required. To address the timing of requirement, we compared Nlg1 null arborisations with those of wild-type controls at 30-36h APF. PM-Mns in Nlg1 nulls at this stage generate similar numbers of very dynamic filopodia compared to controls. A clear difference however was the far greater ‘bendiness’ or the tortuosity of Nlg1 null branches (Figure 4G). To evaluate this, we scored primary and secondary branches of Nlg1 null and control arborisations staged between 30h and 36h APF using an index of bendiness (Figure 4H). This was calculated from the percentage difference between the actual length of branches and the straight-line distance between their nodes. Nlg1 null branches were found to be significantly less straight than those of the controls (Mann-Whitney U = 441, p < 0.0001, twotailed). This early phenotype indicates a requirement for Nlg1 signalling during the very initial phases of pleural neuromuscular development.
Although the full mutants showed robust and consistent phenotypes, there is limitation on what these can tell us. For example, they cannot reveal to us whether the phenotype is due to a failure of ‘local interactions’ between the developing synaptic partners. To address this we generated FLEX-based, FlpStop tools (Fisher et al, 2017) that allow conditional disruption of endogenous Nlg1 expression in a clonal fashion (Figures 5A&B).
Using a MiMIC insertion within the 3rd coding intron of Nlg1 we generated lines capable of either rescuing Nlg1 in a mutant background or mosaically disrupting Nlg1 in a wild-type background. For complete gene disruption, FlpStop lines were used in a heterozygous condition with a deficiency covering Nlg1. To test the ability of the non-disrupting orientation (ND) to disrupt Nlg1 expression upon cassette inversion, hsFlp122 was used to induce large numbers of D-lock clones. At 90h APF, a very similar phenotype was observed to Nlg1 nulls (Figure 5D). To test the ability of the disrupting orientation (D) to be converted into a non-disrupting allele (ND-lock), a germ-line inverted stock was generated. In this case, arbor growth was rescued to a near wild-type phenotype (Figure 5E).
To test the local requirement of Nlg1 we induced small numbers of FlpStop muscle precursor clones that generated fibres containing varying numbers of Nlg1+ nuclei (Figures 5F&H). Looking at stages between 70h and 85h APF, the organisation of arborisations, relative to the pattern of clonal muscle fibres, suggest a local role for Nlg1 mediated adhesion in branch growth. In the initially non-disrupting orientation, terminals growing on non-clonal/low level clonal muscle appear to be phenotypically closer to wild-type (Figure 5G). In contrast, branches belonging to neurons which had grown on muscle with more clones, displayed phenotypes comparable to complete Nlg1 nulls (see Figure 4B). Using the disrupting orientation allele the reverse situation was observed (Figure 5I). Branches growing across clones in which Nlg1 expression was rescued displayed more wild-type morphologies than those growing on non-clonal fibres (n = 8 muscle mosaics). This is demonstrated in Figure 5I by the two arborisations spanning clonal and non-clonal muscles; segments on the wild-type clonal regions show extensive growth, whereas branches on the non-clonal regions show a reduction in growth comparable to complete nulls (n = 7 muscle mosaics).
Dynamic complexes of ‘synaptic’ adhesion proteins stabilise filopodia and drive branch growth
One prediction from these mutant data is that Nlg1 could ‘prepattern’ PM neuromuscular junctions, as is seen with acetylcholine receptors in zebrafish somatic muscles (Panzer et al., 2006). To test this idea we looked at the location of postsynaptic Nlg1 with a GFP tagged version of Nlg1 (Banovic et al., 2010). At 35h APF we found that Nlg1::GFP faintly labels the entire postsynaptic membrane, but forms strong puncta only at sites that directly appose the axon terminals (Figure 6A). To understand what was happening we simultaneously looked at the dynamics of the growing neurons and the localisation of Nlg1. Higher magnification imaging showed Nlg1 puncta were found concentrated on growing branches, particularly at sites of filopodia growth, along the lengths and also at the very tips of filopodia (Figure 6B and movie 7).
To look at the timing of Nlg1 recruitment with regards to branch growth we imaged at 5-minute intervals from 35h APF (Figure 6C). From these time lapses we found that Nlg1::GFP puncta are recruited directly onto filopodia and the tips of growing branches. As a result, branch growth occurs as a highly coordinated sequence whereby the arrival of Nlg1::GFP on exploratory processes precedes their stabilisation and maturation into stable branches in an iterative manner.
The dynamic recruitment of Nlg1::GFP puncta to growing branches and filopodia suggests a significant role for Nlg1 during the earliest phases of arbor construction. To look for a possible relationship between Nlg1::GFP puncta and filopodia/branch dynamics, we took faster time-lapses with 1-minute intervals of arborisations between 30 and 35h APF. Shown in the series in Figure 6D, we found that Nlg1::GFP puncta in apposition to growth cones and filopodia are often stable for many minutes. What is more, these puncta at the tips of filopodia (white arrowheads) appeared to correlate with filopodia longevity. In addition, we found that puncta mark the limits of filopodial retraction (yellow arrowhead). To assess this relationship, we compared the lifetimes of filopodia with and without Nlg1::puncta in time-lapses of 7 individuals. Due to photobleaching, these sequences were limited to ~ 20 minutes. Very few incidents were observed in which newly generated filopodia recruit an Nlg1::GFP punctum. Therefore, instead of analysing newly generated filopodia, only filopodia in existence at the beginning of recording were considered in the analysis. The graph in Figure 6E shows that 55.1% of filopodia not apposed to puncta (n = 49 filopodia) were lost within 10 minutes, 22.5% were lost within 20 minutes and a further 22.5% lasted longer than the duration of the movies. On the other hand, only 4.4% (2 filopodia) of the population apposed to puncta (n = 45 filopodia) were lost within 10 minutes, 2.2% (1 filopodium) were lost within 20 minutes and 93.3% survived for longer than the duration of the recordings. Statistical analysis found that filopodia tipped by Nlg1::GFP puncta are significantly longer lived than filopodia not bearing puncta (Figure 6E).
The major trans-synaptic binding partners of Neuroligins are the Neurexins. Thus, a natural prediction would be that presynaptic Nrx should mirror the postsynaptic distribution of Nlg1::GFP. We expressed GFP tagged Nrx1 (hereon termed Nrx) in the PM-Mns and found that Nrx::GFP (Banovic et al., 2010) forms puncta during early arbor growth that localise to points of filopodial growth and along filopodial lengths, including tips of filopodia (Figure 6F and movie 8). The low signal and rapid bleaching of Nrx::GFP made it difficult to follow Nrx dynamics in vivo over longer periods.
Alongside Nrx we also looked at two key players in presynaptic development; Syd-1 and Liprin-α. Liprin-α (Lar interacting protein) is a scaffolding protein that is one of the first components recruited to trans-synaptic Nrx-Nlg complexes (Owald et al, 2012). Liprin-α::GFP (Fouquet et al., 2009) was expressed in motoneurons with OK371-GAL4. Much like our Nrx::GFP data, Liprin-α::GFP forms distinct puncta at branch terminals and within filopodia (Figure 6G). Similarly to postsynaptic Nlg1::GFP, we found that presynaptic Liprin-α::GFP puncta regularly marked the limits of filopodial retraction (Figure 6H) and appeared to correlate with filopodial stability (see movie 9). Indeed, filopodia bearing Liprinα::GFP puncta were significantly longer lived than those not (Figure 6I). It appears that Liprin-α::GFP coalesces into puncta directly on filopodia (Figure 6J). To ask if Liprin-α::GFP puncta mark adhesion complexes we looked at the localisation of another known interactor, Syd1. Syd1::GFP (Owald et al., 2010), like Liprin-α::GFP, forms puncta which localise within growing branch terminals, including at the tips of filopodia (data not shown). To see if these 2 proteins colocalise to the same sites we expressed Liprin-α::GFP together with Strawberry tagged Syd1 (Syd1::Straw) (Owald et al., 2010). The large majority of puncta of each protein were coincident, including those at the tips of filopodia (Figure 6K). In contrast, when Liprin-α::GFP and BRP::RFP were expressed together there was very little co-localisation at branch points and never at filopodia tips (Figure 6L).
Finally, to determine the subcellular distribution of Liprin-α::GFP in the central nervous system we looked at Eve+ interneurons. Like in growing axon terminals of PM-Mn neurons, Liprin-α::GFP is localised to the tips and along the lengths of filopodia on the growing axonal output arborisations (Figure 6M).
Nlg1-based adhesion complexes can direct a tropic mode of growth
The relationship between Nlg1::GFP puncta and filopodial dynamics suggests that it plays a role in the growth of axonal arborisations by providing adhesive stability to branches and filopodia during the early stages of growth. A direct prediction from this would be that if we manipulated the postsynaptic levels of Nlg1 at early stages we would see changes in PM-Mn growth. To explore this, we used UAS-Nlguntagged which is known to be expressed at high levels and cause a strong phenotype in larvae (Banovic et al., 2010).
At 33-35h APF, elevated postsynaptic Nlg1 levels resulted in compacted axon terminals with distal branches transformed into flattened growth cones with many filopodia (Figures 7A&B). This irrevocable effect on growth led to late stage arborisations with reduced branch length, territory and complexity (Figures 7C&D). These branches also maintained greater than usual numbers of filopodia into the later stages of development.
Although full postsynaptic overexpression of Nlg1untagged revealed that axon terminals are sensitive to Nlg1 levels from very early stages of growth, to explore how different levels of Nlg1 signalling impacts branch growth at a local level we developed a clonal technique that generates patterns of expression much like a ‘Bonhoeffer stripe’ assay (Walter et al., 1987). With this ‘stripe assay’ we can present growing motoneurons with different levels of Nlg1untagged to grow upon (Figure 7E). At early stages (35h APF), PM-Mn growth was directed onto myoblast clusters/developing myotubes that strongly expressed the Nlg1untagged (Figure 7F). By later stages (70h APF), branches in contact with these strongly expressing clonal fibres displayed the same hyper-stabilisation phenotype seen with full muscle expression. In contrast, branches from the neuron in contact with low to non-expressing fibres grew as normal, demonstrating unequivocally that Nlg1 impacts branch growth via local mechanisms (Figure 7G). Although branches contacting highly expressing fibres had reduced growth and branching, they could sometimes be seen to elaborate along a clonal fibre, suggesting a ‘tropic’ mode of growth is at play.
To further investigate Nlg1 signalling on branch growth we took advantage of the organisation of the peripheral nervous system in the abdominal body wall. As shown in Figure 7H, the class IV dendritic arborisation sensory neuron v’ada elaborates on the internal side of the epidermal cells. The peripheral neurites of v’ada are separated from the motor axon branches by the pleural muscles, which in turn receive innervation from the PM-Mns onto their internal surface. During early pupal development, motor and sensory arborisations are in very close proximity. We predicted that if we expressed Nlg1 in these usually ‘asynaptic’ sensory neurites the signalling would direct the growth of the PM-Mn arborisations into a novel territory. We expressed Nlg1untagged in v’ada neurons and then visualised the anatomy of the motoneurons. In controls branch growth was restricted exclusively to the inner surface of the muscle (Figures 7I&J). As highlighted by the transverse projection there is a relatively uniform layer of motoneuron terminals (Figure 7K). In contrast, when v’ada was made to misexpress Nlg1untagged, PM-Mn axon branches grow perpendicular to the normal arborisation between muscle fibres to contact v’ada sensory neurites (Figures 7L-N). In several cases further branching from these contacts increased the complexity of these ectopic PM-Mn branches.
One postulate of Vaughn’s tropic model of arborisation growth was that the stabilisation of contacts between synaptic partners would ultimately lead to functional connectivity (Vaughn, 1988). For this to be satisfied it would be expected that branches generated by such a mechanism would harbour mature synaptic terminals later in development. To test this in branches sculpted by ectopic Nlg1untagged, abdominal fillets were stained for the active zone marker BRP using the antibody NC82 and with the neural marker, anti-HRP. As shown in Figure 7O, conspicuous concentrations of BRP were found at each contact. Alongside this, we also found strong VGlut immunoreactivity at these ectopic synaptic contacts (data not shown).
Physiological consequences of Nlg1 disruption
We have shown a very early requirement for Nlg1 for establishing branch structure. The ectopic contacts that PM-Mns make on v’ada neurites appear to mature into hemisynapses. These data show that following contact and stabilization there is a hierarchy of events that ultimately leads to synapse formation. To explore if Nlg1-Nrx disruptions impact synapse formation we stained against BRP to assess the density of active zones within the branches of Nlg1 nulls, Nrx nulls and animals expressing Nlguntagged in muscles at pupal stages close to eclosion (Figure 8A). Boutons in all these animals were considered as engorgements containing concentrations of synaptic puncta. We found that the density of synapses was not significantly different between Nlg1 nulls or Nrx nulls and wild-type controls, although it was slightly higher in Nlg1 gain-of-function animals (Figure 8B). This suggests that global aspects of active zone development may be largely unaffected by loss of Nlg1 or Nrx. One might expect that functional deficits arising from a failure in appropriate Nlg1 signalling could be due to either defective morphology and to changes in the transmission or both. To explore this, we looked at GCaMP6m expressed in the muscles as a proxy for measuring muscle depolarisation at late pupal/pharate stages just prior to eclosion. In wild-type controls, muscle calcium events at this stage usually occur in synchrony across the muscle field (Figures 8C&D and movie 10). In contrast, in Nlg1 gain-of-function pupae, calcium events were more common in some groups of fibres than others, resulting in far less synchronicity (Figures 8E&F and movie 11).
To determine the functional consequence of disruption to Nlg1-Nrx signalling, we also assessed locomotor ability of Nlg1 null flies in a climbing assay (Figure 8G). Using videography and an automated tracking software we found that the climbing speed of Nlg1 nulls (n = 11) was significantly lower than controls (n = 10) (Figure 8H) (t(8.15) = 19, p < 0.0001, t-test, two-tailed). We found the leg motoneurons disrupted in Nlg1 nulls, suggesting that some of this deficit may be the result of either disrupted growth of leg motoneurons arborisations or in central circuits (Figure 8I).
Discussion
PM-Mn axonal arborisations use a dynamic ‘synaptotropic-like’ mode of growth
Building arborisations of the right size and shape is critical for proper neural circuit development and function. Live imaging studies in vertebrate brains show that neuronal growth is highly dynamic and that structures which appear to be nascent synapses play a key role in the development of axonal and dendritic arborisations. These nascent contacts are believed to act like bolts during construction, yet a detailed understanding of their molecular composition and assembly is not known.
To explore this biology, we searched the fly for neurons that grow similarly to those in the fish and frog visual systems. Although fly embryos have been an excellent tool for studying connectivity, the small size and rapid development of their axonal arborisations makes it very challenging to image their growth live.
The axonal arborisations of the pleural muscle motoneurons (PM-Mns) are superficial and can be imaged in vivo throughout metamorphosis. Unlike the da sensory neuron input arborisations, which have been useful for studying neurite branch growth (Williams & Truman, 2004), PM-Mn axons form synapses; a trait in common with the majority of neuronal arborisations.
PM-Mn growth is very dynamic with a high turnover of exploratory filopodia, only a few of which ultimately become branches. Arborisations develop in close association with the pleural muscles and we find that axonal filopodia are stabilised upon contact with myoblasts or immature myotubes (see movie 2). A number of in vitro and in vivo studies have previously highlighted that filopodial stability is conferred by the contacts made between potential synaptic partners (Cooper & Smith, 1992; Ziv & Smith, 1996; Jontes et al., 2000).
To determine whether PM-Mn axonal arborisations could be growing using nascent synapses we imaged the active zone marker Bruchpilot (BRP) (Chen et al., 2014; Urwyler et al., 2015) and vesicular machinery (VGlut and Syt1) and found similar localisations of synaptic proteins, as well as relationships with branch lifetimes as have been described in zebrafish and Xenopus (Alsina et al., 2001; Niell et al., 2004; Meyer & Smith, 2006; Ruthazer et al., 2006). BRP puncta appear to chart the progression of branch stabilisation events. To our surprise we also found Bruchpilot at similar localisations in the branches of developing eve+ interneurons in the CNS, indicating that this type of growth may be common within the fly nervous system.
Growing arborisation do not require functional synapses
The exact contributions of synaptogenesis, neural activity and synaptic transmission to the formation of neural networks are unclear. Indeed, there is a rich history regarding the question of whether nervous systems develop in “forward reference” to, but without benefit from, functional activity (Harrison, 1904, Weiss,1941; see Haverkamp, 1986 for review).
Interestingly, we find that a great deal of PM-Mn outgrowth takes place prior to robust presynaptic calcium transients (action potentials, <42h APF). Furthermore, not until 60h APF could we evoke activity in muscles by stimulating the motoneurons. Finally, neither TTX or removing all vesicular neurotransmission, by making VGlut null clones, had an overt impact on mature arbor morphology. It may be that different systems are intrinsically different; Cline and colleagues showed a correlation between the stabilisation of growing retinal ganglion cell axon branches bearing synaptic puncta and visual activity, pointing to use-testing of nascent contacts by activity in a synaptotropic-like mode of growth (Ruthazer et al., 2006). In contrast however, a number of studies have shown that blocking activity has little impact on morphology, or plays only a role in the refinement of arbor growth (Haverkamp, 1986, Verhage et al., 2000; Varoquoaeux et al., 2002; Hua et al., 2005; Fredj et al., 2010).
The detailed imaging possible with our system reveals that BRP puncta very rarely enter filopodia and are never stabilised within them. We found the same to be true for the synaptic vesicle associated proteins Syt1 and VGlut. Since BRP is an important structural component of the cytomatrix at the active zone, these data seemed at odds with the notion that synapses drive branch stabilisation and led us to question if puncta previously reported at branch nodes in growing retinal ganglion cells really represent genuine synapses. Alongside this, we found it was not until late stages of development that the size and distribution of BRP puncta became more akin to synapses at the larval NMJ (Fouquet et al., 2009). In parallel, we saw conspicuous clusters of glutamate receptors only at late stages, long after arbor shape has been established. Taken together these data suggest that it is only at the later stages of arbor development that synaptic elements pair to form bona fide synapses. The accumulations of presynaptic proteins seen in early arborisations (i.e. 30-40h APF) may instead mark stable sites or ‘transport hubs’ that help store/sort synaptic proteins for a later role in synapse formation.
Neuritic adhesion complexes (NACs) drive a tropic mode of arborisation growth
If synaptic transmission does not play a role in this type of dynamic arbor growth, what mechanisms are responsible? We find a role for a class of proteins (synaptic cell adhesion molecules), pre-synaptic Neurexins (Nrxs), and their postsynaptic binding partners, Neuroligins (Nlgs).
The Nlg1 mutants and the clonal FlpStop data suggest that Nlg1 in the muscle targets is crucial for normal growth. Clonal analysis reveals that branches growing onto muscles with lower levels of Nlg1 expression are phenotypically similar to those in Nlg1 nulls. The growth of branches onto territories with more wild-type levels of Nlg1 expression is reminiscent of the tropic aspect suggested in Vaughn’s original hypothesis.
Work in Xenopus has previously implicated Nrx-Nlg interactions in arbor growth and posited that synapse formation and subsequent levels of synaptic transmission translates directly into branch stability (Chen et al., 2010). Here we propose that these molecules provide adhesion during elaborative phases independent of synapse formation. We describe for the first time that Nlg1::GFP puncta emerge on muscles in vivo following contact with a presynaptic filopodia. Nlg1 localises onto the tips and shafts of filopodia and regulates stability. Filopodia that ‘capture’ such puncta are significantly longer lived than those that do not. Furthermore, time-lapse imaging revealed that Nlg1::GFP act like anchor points by marking the limits of filopodial retraction. This is very different to the growth of zebrafish and mouse neuromuscular junctions, where motoneurons grow between pre-patterned plaques of acetylcholine receptors, as if hopping between stepping stones ((Yang et al., 2001). Panzer et al., 2006; Jing et al., 2009).
Presynaptically we found Nrx puncta on the tips of filopodia and along their lengths i.e. at sites that mirror those of Nlg1. The two other proteins we also find at such sites are the presynaptic membrane associated proteins Syd-1 and Liprin-α, both of which are known to complex with Nrx. Liprin-α and Syd-1 are found on the membrane and coalesce into dynamic puncta that localise to filopodia tips. Liprin-α puncta, appear to limit filopodial retraction, indicating that sites marked by this protein represent the presynaptic counterparts to the structural anchor points marked by Nlg1.
Previously, Syd-1 and Liprin-α have been found to play a key role in orchestrating synapse assembly by recruiting and retaining other synaptic proteins. Liprin-α, Syd-1 and Nrx puncta at these stages of exuberant growth are transient and do not appear to become ‘future synapses’. Their fluidity speaks to a role in branch morphogenesis rather than in a stepwise, clockwork assembly of synaptic machinery at a particular place.
Based on our observations, we propose that these dynamic complexes are composed of a subset of proteins that have been previously called ‘synaptic cell adhesion proteins’ or drivers of synapse formation. As a placeholder we suggest calling these puncta neuritic adhesion complexes or NACs to highlight their role in axon growth prior to synapse formation. That we see direct evidence for the formation of these NACs on filopodia themselves suggests that dynamic interactions between filopodia and the postsynaptic target are fundamental determinants of tree construction.
This early role for Nlg1/Nrx based NACs in arbor growth is supported by strong evidence that exploratory filopodia and branch tips are highly sensitive to changes in the early expression of postsynaptic Nlg1. Our gain-of-function myoblast/myotube stripes of clones seem to have a ‘hyper-stabilising’ effect on wild-type neurons and ultimately constrain growth. Moreover there is some indication of preferential growth into regions with elevated postsynaptic Nlg1 expression. Remarkably, we were very surprised to see wildtype PM-Mns grow past muscles, into novel territories and make contacts with class IV da sensory neurons ectopically expressing Nlg1.
Physiological consequences of Nlg1 disruption
The precise role that Nrxs and Nlgs play in nervous system development has been broadly disputed. Previous work has shown the strong synaptogenic potential of these proteins by expressing them in HEK cells or on micropatterned substrates revealing them to be potent regulators of hemisynapse formation (Scheiffele et al., 2000; Dean et al., 2003; Graf et al., 2004; Chih et al., 2005; Lee et al., 2010; Czöndör et al., 2013). Others show that mice lacking three Nlg homologues or all three α-Nrxs build brains with grossly normal cytoarchitectures and synapse densities (Missler et al., 2003; Varoqueaux et al., 2006) and posit that their role is solely to modulate synapse function. Roles for Nlg and Nrx in synapse formation have previously been described in the fly (Li et al., 2007; Banovic et al., 2010; Chen et al., 2012).
Teasing apart the function of such gene families in vertebrates can been difficult due to the multiple copies of these genes and their degeneracy with other proteins (Sugita et al., 2001; Ko et al., 2009; Siddiqui et al., 2010; Xu et al., 2010; Ko et al., 2011; Soler-Llavina et al., 2011). Here in this simpler system we show that Nlg1/Nrx based adhesion complexes play a key role in the early growth of axonal arborisations prior to the formation of functional synapses. In addition, we find that disrupting Nlg1/Nrx signalling has little effect on the timing of development and size of active zones, indicating that tree morphogenesis is uncoupled from the formation of synapses. One prediction from this is that disruption in functional connectivity would largely be due to disruptions in growth and morphology of the arborisation. An important observation that frames this is the outcome from ectopically expressing Nlg1 in class IV da sensory neurons. There we see the generation of a ‘synthetic connectivity’ on the normally ‘asynaptic’ da sensory neurites with the development of mature, differentiated presynaptic structures in later pupal stages i.e. synaptic vesicles and BRP labelled synapses.
In support of this, our calcium imaging reveals that the normal patterns of muscle activity are disrupted when Nlg1 is overexpressed, with changes in the frequency and spatial heterogeneity of calcium. Since elevated postsynaptic Nlg1 expression results in condensed arborisations, we propose that this is a result of irregular innervation. Additionally, the behavioural analysis shows that Nlg1 null adults had significant motor deficits. Taken together these data provide a link between an adhesion-based mechanism of arbor growth and synaptic connectivity which ultimately effects function.
Conclusions
In this study we have demonstrated, for the first time outside of vertebrates, a dynamic ‘synaptotropic-like’ mode of arbor. In contrast to the mechanisms that have previously been proposed, we find no evidence that functional synapses drive this type of growth. Instead, our data point to dynamic NACs, composed of Neurexin, Neuroligin, Liprin-α and Syd-1, being important for stabilising filopodia during arborisation growth. Alongside this, our evidence showing the same subcellular localisations in Drosophila central neurons points toward this mode growth being a universal mechanism for building complex trees. It may be that Berry and colleagues’ emphasis on filopodia in their ‘synaptogenic filopodial theory’ was correct. It is an appealing idea that such mechanisms could construct axonal and dendritic arborisations of a broad spectrum of shapes and sizes by making only subtle changes to a ‘stick and grow’ algorithm. Exactly which proteins constitute these adhesion complexes and how they interact with the machinery for regulating cytoskeletal remodelling will be a focus of future investigation.
Materials and Methods
Fly stocks
Flies were reared on a standard yeast-cornmeal-molasses diet. For time-sensitive experiments flies were raised at 25°C, or at room temperature if precise staging was not required.
OK371-GAL4; UAS-myr::GFP
OK371-GAL4; UAS-myr::GFP, UAS-BRP::RFP
OK371-GAL4; UAS-Lifeact::Ruby/UAS-CLIP170::GFP
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Mef2-GAL4, UAS-mCD8::Cherry
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Mef2-GAL4, UAS-myr::tdTomato
OK371-GAL4, UAS-mCD8::Cherry/BRP::GFP
OK371-GAL4, UAS-mCD8:Cherry/VGlut::GFP
OK371-GAL4, UAS-mCD8:Cherry/Syt1::GFP
RN2-Flp, Tub-FRT-CD2-FRTGal4, UAS-mCD8GFP/UAS-BRP::RFP
OK371-GAL4, UAS-mCD8:Cherry/GluRIIE::GFP OK371-GAL4, UAS-GCaMP6m
VGlut(Trojan)-T2A-LexA; Mef2-GAL4, UAS-GCaMP6M/LexAop-TRPA 1
VGlutNMJX-Gal4, UAS-mCD8::GFP, hsFlp122; FRT40A, Df(2L)VGlut2; UAS-myr::GFP
VGlutNMJX-Gal4, UAS-mCD8::GFP, hsFlp122; FRT40A, Df(2L)VGlut2/FRT40A; UAS-myr::GFP
Elav-GAL4, hsFlp122; FRT40A, Df(2L)vglut2/FRT40A; UAS-cytoplasmicGFP
VGlut(Trojan)-T2A-LexA, LexAop-myr::tdTomato; Mef2-GAL4, UAS-Nlg1::GFP
UAS-Nrx::GFP; OK371-GAL4, UAS-mCD8::Cherry
OK371-GAL4, UAS-mCD8::Cherry/UAS-Liprin-α::GFP
OK371-GAL4, UAS-Liprin-α::GFP/UAS-Syd 1::Straw
OK371-GAL4, UAS-Liprin-α::GFP; UAS-BRP::RFP
RN2-Flp, Tub-FRT-CD2-FRT-GAL4, UAS-mCD8::GFP/UAS-Liprin-a::GFP
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Df(3R)BSC747/Nlg1ex2.3
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Df(3R)BSC747/Nlg11960
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Df(3R)Exel6191/Nrx241
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Mef2-GAL4/UAS-Nlg1untagged
hsFlp122; VGlut(Trojan)-T2A-LexA, LexAop-myrGFP, αTub84B-FRT.GAL80; Mef2-GAL4, UAS-myr::tdTomato/UAS-Nlg1untagged
PPK-GAL4, UAS-dsRed/VGlut-LexA, LexAop-myr::GFP
PPK-GAL4, UAS-dsRed/VGlut-LexA, LexAop-myr::GFP; UAS-Nlg1untagged
PPK-GAL4, UAS-mCD8::GFP; UAS-Nlg1untagged
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Nlg1FlpStop D/Df(3R)BSC747
VGlut(Trojan)-T2A-LexA, LexAop-myrGFP; Nlg1FlpStop ND/Df(3R)BSC747
hsFlp122; VGlut(Trojan)-T2A-LexA, LexAop-myrGFP/Mef2-GAL4; Nlg1FlpStopND/Df(3R)BSC747
hsFlp122; VGlut(Trojan)-T2A-LexA, LexAop-myrGFP/Mef2-GAL4; Nlg1FlpStopD/Df(3R)BSC747
Mef2-Gal4, UAS-GCaMP6m; UAS-Nlg1untagged
Mef2-Gal4, UAS-GCaMP6m
Imaging
Imaging was performed at room temperature using Zeiss LSM 510 or 800 series confocal microscopes with EC Plan-Neofluar 20x/0.50 or Plan-Apochromat 40x/1.3 objectives, except the laser ablation experiments, which were performed using a Nikon A1R with an Apochromat 40x/1.25 objective.
Mounting and live imaging
For consistent developmental staging, pupae were collected at 0h APF (white prepupal stage) and incubated on moist tissue paper at 25°C in parafilm sealed petri dishes. In preparation for mounting, pupae were dried on tissue paper, immobilised on strips of double sided sticky tape and carefully removed from their puparial cases using forceps. Pupae were mounted using 22x22mm cover slips beneath a thin coating of halocarbon oil. For imaging sessions of greater than 1-hour pupae were mounted in humidity maintained chambers consisting of a platform of semi-permeable membrane suspended above a hole in a specialised steel slide and sealed with a watertight ring of petroleum jelly.
For experiments requiring temperature manipulation, a specialised temperature adjustable stage was built consisting of a glass slide fastened to a 5x5cm thermoelectric Peltier device (Maplin Electronics Ltd., UK) which was mounted on a regular microscope slide-holder. Before each experiment the temperature of the stage was calibrated using a thermocouple probe attached to a multi-meter device (Rapid Electronics Ltd., UK) and controlled from a 0-30v power supply unit.
Mosaic analysis
The MARCM method was used to generate and label VGlut null motoneuron clones. A preliminary MARCM screen using OK371-GAL4 found that the pleural muscle motoneurons are born during the embryonic wave of neurogenesis. To generate clones during this wave of mitosis, breeding adults were allowed to lay on grape jelly plates with yeast for 2 hours at 25°C. Eggs were incubated for 3 hours at 25°C and heat shocked by incubation in a water bath at 37°C for 45 minutes, followed by resting at room temperature for 30 minutes, followed by a further 30 minutes at 37°C. Upon hatching, larvae were transferred to standard food and raised at 25°C.
To generate small numbers of GAL80 ‘flip-out’ or FlpStop Mef2-GAL4 positive muscle clones L3 wandering larvae were heat shocked for 20 minutes by incubation in a water bath at 37°C.
Dissections and immunocytochemistry
Dissections were made in Sylgard silicone elastomer (Dow Corning, USA) lined petri dishes in 1x phosphate buffered saline (PBS) (pH 7.3, Thermo Fisher Scientific, USA).
For dissections of pupal abdominal body walls, pupae were removed from their puparial cases and pinned via the head using electrolytically sharpened 0.1mm tungsten pins. The posterior tip of each abdomen was removed (from approximately segment 6) and an incision made along the dorsal midline until reaching the thorax. Abdomens were separated from the thorax by cutting along the joint and laid flat using additional tungsten pins. Viscera were removed by a combination of gentle pipetting and forceps. Larval body wall dissections were performed as described previously (Broadie & Bate,1993)
Dissected samples were fixed in buffered 3.6% formaldehyde for 45 minutes at room temperature and washed three times in PBS containing 0.3 Triton-X100 (Sigma, USA) (PBST). Following fixation, samples were blocked in PBST containing 4% goat serum (Sigma, USA) for 45 minutes and incubated in solutions of primary antibodies made up with PBST overnight at 4°C. After rinsing 3 times with PBST over the course of a day, samples were incubated in solutions of secondary antibodies made up with PBST overnight at 4°C. After 3 rinses with PBST and a final rinse with PBS, samples were mounted on poly-L-lysine coated coverslips, dehydrated through a series of alcohol, cleared with xylene and mounted in DePeX (BDH Chemicals, UK).
Primary antibodies were used at the following concentrations: mouse anti-NC82 (DSHB, USA) 1:5, rabbit anti-VGlut-C term (Gift from Hermann Aberle (Mahr & Aberle, 2006)) 1:10,000, chicken anti-GFP (Invitrogen, USA) 1:1000. Secondary antibodies were used at the following concentrations: Cy3 goat anti-HRP (Jackson Immunoresearch, USA) 1:5000, AlexaFluor 488 goat anti-Chicken (Invitrogen, USA) 1:500, Cy5 donkey anti-mouse 1:500, Cy5 donkey antirabbit 1:500.
For dissections of CNSs, white pre-pupae were collected and incubated at 25°C. Pupae were dissected from their puparial cases as described above. CNSs were dissected out in 1x PBS in Sylgard lined Petri-dishes. These were then immediately fixed in 3.6% formaldehyde for 20 minutes. Fixative was washed off thoroughly using 1x PBS. CNSs were mounted on glass slides in Slow-Fade Antifade reagent (Thermo Fisher Scientific) and sealed beneath coverslips using nail-varnish.
Microinjections
To prepare for injection, pupae were dried on tissue paper and secured to glass slides using double sided sticky tape. Needles, pulled from glass capillaries, were loaded with 0.5mM TTX made up in PBS or a control solution of PBS. Injections were made into the abdominal cavities through small access holes made in the puparial cases, using a Transjector-5246 micro-injector (Eppendorf, Germany). Following injection, animals were removed from the sticky tape with a damp paintbrush and incubated at 25°C.
Climbing assay
Adult females were used two days post-eclosion. To assess climb speed, each fly was placed in a square plastic tube with 1 cm edges and transparent walls. The narrow calibre prevented flies from jumping or flying. Each fly was tapped to the bottom and its climb recorded with an S-PRI high-speed camera (AOS Technologies AG, Switzerland) at 700 fps, frame size of 900x700 pixels, under red light illumination. Automated body tracking was performed using FlyLimbTracker (Uhlmann et al., 2017) in Icy (de Chaumont et al., 2012) over a distance of 0.27 to 0.8 cm. Only recordings in which the climbing path had a linearity over 90%, as determined with the Motion Profiler processor in Icy, were considered for analysis. The average climb speed of each fly was determined by calculating the displacement of the centroid of the body between each frame over 3 repeats.
Image Analysis
Raw image stacks were imported into the image processing software Fiji (http://imagei.net/Fiii) for enhancement of brightness and contrast. For clarity, the freehand select tool was used to remove obscuring objects created by degenerating larval tissues and macrophages on a slice by slice basis. Collapsed z-projections were imported into Photoshop (Adobe, USA) for figure assembly. Schematic cartoons were made in Photoshop or Illustrator (Adobe, USA). To generate kymographs the re-slice tool in FIJI was used to reconfigure the axis of image sequences.
Statistical analysis
Statistical analyses were performed using the software package GraphPad Prism (GraphPad Software Inc., USA). Significances correspond to the following p values: <0.05 = *, <0.01 = **, <0.001 = ***, <0.0001 = **** For selecting between parametric and non-parametric tests, the residuals of each data set were tested for normality. Results are reported with standard deviations (SDs).
Quantification of synaptic puncta
Relationships between BRP::RFP, Nlg1::GFP and Liprin-α::GFP puncta dynamics and branch growth were calculated by manually counting and tracking puncta in FIJI. Puncta were identified by accumulations of fluorescent protein measuring 3 or more pixels in diameter at the native resolution. The diameters of BRP::RFP puncta were measured by intersection with the line tool in FIJI at their widest point and calculation of the full width at half maximum of the peak in fluorescence.
Morphometric analysis
Arbor reconstructions were generated using the semi-automated plugin for FIJI, Simple Neurite Tracer. Reconstructed skeletons were imported into a bespoke analysis software in which total arbor length, total branch number, Strahler order and total arbor were computed. Arbor area was calculated by giving branch coordinates a hypothetical ‘contact distance’ which represented its area of coverage. This value was set to 20μm. In addition, this software could be used to add and delete vertices and nodes, allowing corrections to me made to tracing errors.
Straightness Index
To calculate branch bendiness, the ‘actual’ length of primary, secondary and tertiary axon branches was measured using the freehand line tool in FIJI as the distance between their nodes (or in the case of primary branches, between nodes and tips). These values were divided by the direct distances between these nodes, measured using the straight-line tool, giving an index of bendiness.
Active zone density
Active zone densities in axon terminals were calculated manually in FIJI using the multi-point tool. Bouton areas were calculated by tracing around boutons using the freehand selection tool.
Acknowledgements
We would like to thank Hermann Aberle for providing the Nlg1 alleles and UAS reagents as well as the OK371-GAL4 enhancer-trap line and anti-VGlut antibodies, Stephan Sigrist for providing the Liprin-α and Syd1 reagents, Barret Pfeiffer for providing the UAS-GFP lines with translational enhancers, Aaron DiAntonio for sharing the Df(2L)VGluŕ allele, Frank Schnorrer for providing a copy of Mef2-GAL4, Vijay Raghavan for sending the FlyFos GluRIIE::GFP line and Tom Clandinin for kindly sharing his FlpStop construct and for advice on implementing it. In addition, we thank Jon Clarke for generously allowing us to use his confocal microscope. We also thank Matthias Landgraf for providing the VGlut-LexA, discussions and advice on experiments. Finally, we thank Jon Clarke, Matthias Landgraf, Martin Meyer and Laura Andreae for reading the manuscript. This work was funded by the BBSRC.