Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially

Dominant mutations in CACNA1A, encoding the α-1A subunit of the neuronal P/Q type voltage-dependent Ca2+ channel, can cause diverse neurological phenotypes. Rare cases of markedly severe early onset developmental delay and congenital ataxia can be due to de novo CACNA1A missense alleles, with variants affecting the S4 transmembrane segments of the channel, some of which are reported to be loss-of-function. Exome sequencing in five individuals with severe early onset ataxia identified one novel variant (p.R1673P), in a girl with global developmental delay and progressive cerebellar atrophy, and a recurrent, de novo p.R1664Q variant, in four individuals with global developmental delay, hypotonia, and ophthalmologic abnormalities. Given the severity of these phenotypes we explored their functional impact in Drosophila. We previously generated null and partial loss-of-function alleles of cac, the homolog of CACNA1A in Drosophila. Here, we created transgenic wild type and mutant genomic rescue constructs with the two noted conserved point mutations. The p.R1673P mutant failed to rescue cac lethality, displayed a gain-of-function phenotype in electroretinograms (ERG) recorded from mutant clones, and evolved a neurodegenerative phenotype in aging flies, based on ERGs and transmission electron microscopy. In contrast, the p.R1664Q variant exhibited loss of function and failed to develop a neurodegenerative phenotype. Hence, the novel R1673P allele produces neurodegenerative phenotypes in flies and human, likely due to a toxic gain of function.

Introduction Voltage-gated calcium channels (VGCCs) are a family of calcium ion selective proteins that both mediate calcium entry into neurons at synapses upon depolarization and are required for calcium-dependent functions in the cell [1,2]. VGCCs are composed of multiple subunits, including the α1 subunit which is encoded by the CACNA1A gene and is responsible for Ca 2+ entry in neurons. Across species the α1 subunit is required in the nervous system. In mice the tottering (tg) mutants have mutations in Cacna1a and exhibit ataxia, motor seizures and cerebellar degeneration [3]. In Drosophila the channel is required for synaptic transmission and cacophony (cac) null alleles are lethal [4,5]. In mosaic clones mutations in cacophony produce neurodegeneration with defective lysosomal fusion with autophagosomes [6].
Given the essential role of VGCCs in the nervous system it is not surprising that mutations in CACNA1A cause a spectrum of neurological disorders in humans [7]. Spinocerebellar ataxia type 6 (SCA6, OMIM #183086) is characterized by ataxia, dysarthria, and dysphagia with onset typically in middle adulthood but ranging from 20 to 60 years of age [8]. SCA6 is due to heterozygous expansions of CAG repeats within CACNA1A resulting in large polyglutamine tracts within the protein which cause cytoplasmic aggregations and Purkinje cell degeneration [9,10]. Episodic ataxia type 2 (EA2, OMIM #108500) is characterized by episodes of ataxia with onset in childhood or early adulthood, and affected individuals are often responsive to carbonic anhydrase inhibitors such as acetazolamide [11][12][13]. EA2 is due to heterozygous deletions, stop-gains, frameshifts, or missense mutations [11,14,15]. Functional analysis based on patch-clamping suggests that they correspond to loss of channel function and hence reveal haploinsufficiency of the locus [16,17]. While EA2 is typically episodic, many patients also have progressive ataxia due to ongoing neurodegeneration which is generally slow [15]. Familial hemiplegic migraine (FHM, OMIM # 141500) is a form of migraine with aura and transient hemiplegia and an age of onset between 5 years and early adulthood [18]. CACNA1A variants associated with FHM are typically missense alleles [19], and in contrast to EA2-associated missense alleles, electrophysiological evidence suggests that they are gain-of-function mutations [20]. FHM mutations appear to lead to hyperactivity of the channel [21] both by altering the biophysical properties as well as decreasing the inhibitory G-protein association with the channel [22].
While the disease phenotypes for EA2 and FHM appear to relate to different underlying genetic mechanisms, considerable phenotypic overlap between FHM and EA2 means that some individuals exhibit features of both disorders [23,24]. Indeed even within the same family, the same CACNA1A variants can produce phenotypes more similar to FHM than EA2 [23,25]. Several de novo missense alleles in CACNA1A have been reported in children with congenital ataxia and intellectual disability [26], as well as with non-progressive congenital ataxia with seizures [27]. However, channel function and its relation to phenotype are not well studied in these severe ataxias [26][27][28]. Most previous studies used in vitro electrophysiological analyses to assess channel function [20,21,29], leaving a need for functional annotation of CACNA1A and its variants in a model organism.

Human subjects
We ascertained 5 individuals who underwent exome sequencing for global developmental delay and congenital ataxia, in whom de novo missense variants in CACNA1A were discovered. Patient 1 was enrolled in the Undiagnosed Diseases Network (UDN) at Baylor College of Medicine and concurrently in the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) [30] as part of a large-scale research re-analysis of clinical exomes [31]. Patients 2-5 were identified at Baylor Genetics Laboratories (BGL) [32,33]. All families gave written consent for exome sequencing.
The clinical findings of these individuals are summarized in Table 1 (S1 Case Histories). All individuals (5/5) exhibited global developmental delay, expressive language delay and dysarthric (4/5) or no expressive speech (1/5; Patient 1). Interestingly all subjects had ataxia (5/5) but to varying degrees, with independent ambulation and unsteady gait in some (3/5) and more severely impaired ambulation requiring use of walker in others (2/5). Other neurological features such as behavioral problems, sensory processing disorders, aggressive behavior and attention deficit were also noted, although these features were not consistent among the patients.
Neuroimaging for the patients differed; one of the five subjects had evidence of progressive cerebellar atrophy. In Patient 1, the initial MRI at 10 months showed a normal cerebellum (Fig  1A), while imaging at 22 months revealed mild cerebellar atrophy (Fig 1B), which progressed at 3.5 years (Fig 1C) and 8 years (Fig 1D). Patient 1 is the only subject with progressive cerebellar degeneration. In Patient 3, the cerebellum appeared normal in size at 2 years, although the corpus callosum was thin posteriorly (Fig 1E). In Patient 5 the MRI is normal (Fig 1F). In Patient 4 there is some cerebellar hypoplasia involving the vermis (S1A   Fig). Notably, the four individuals with p.R1664Q and the single individual with p.R1673P all exhibit some similarities to a child previously reported with a de novo p.R1664Q allele with early onset ataxia without seizures or migraine [34].
For each case the ratio of variant reads to total reads and the Sanger confirmation suggested approximately 50% variant alleles meaning the patients are heterozygous. Both de novo changes occur within CpG dinucleotides, hypermutable sites prone to methylation and deamination leading to de novo events [35][36][37]. These have been noted to affect arginine residues in a number of disease contexts [38][39][40]. The de novo missense changes in these patients affect conserved arginine residues at the S4 transmembrane segment of domain IV of the protein (Fig 2A and 2A'). Several pathogenic alleles associated with a range of phenotypes are reported to affect this transmembrane segment of domain IV in ClinVar [41] (Fig 2A'). For example, p. R1661H is associated with EA2, while p.R1667W is associated with FHM. The R1664Q allele showing an approximately mid-sagittal image of Patient 1 at 10 months of age. The cerebellum appears seen in our patients 2-5 is associated with ataxia and global developmental delay. We observed that the intervals of the transmembrane domains (I-IV) all appear intolerant to missense variation (Fig 2B).

Drosophila cac mutants and Drosophila transgenics
Notably these S4 arginine variants in domain IV display diverse phenotypes, and other severe cases reported also carry missense variants within the S4 segments in domains III [26,27,42] and IV [34] of CACNA1A. However, these mutations have not been modeled in vivo, and the functions of these missense variants are therefore not defined (i.e., haploinsufficient loss or gain of function). As drugs are available to either boost or inhibit calcium channel function, knowledge of mutation mechanism may have important implications for patients. In Drosophila cacophony (cac), the homolog of CACNA1A, is required for synaptic transmission and lysosomal fusion, and cac null alleles are embryonic lethal [4,5]. Previously, we isolated numerous alleles of cac in a forward genetic screen for essential genes that affect the function of photoreceptors based on defective ERGs in homozygous mutant eye clones [20]: cac J is an early nonsense mutation which is an embryonic lethal, and cac F is a missense mutation affecting a key glutamate residue in the calcium ion selectivity filter loop and is larval lethal (Fig 3A and 3B).
To test the functional consequences of the variants identified in the five subjects, we designed a rescue-based strategy. To rescue the phenotypes associated with the cac alleles, we first selected a 77 kb P[acman] transgenic construct [6] that contains the entire 53 kb genomic region of cac, including endogenous enhancers to drive proper expression of the transgene. We introduced the two variants found in the subjects by recombineering and we inserted these genomic rescue (GR) constructs into the identical VK37 docking site [6] in the fly genome by phiC31-mediated recombination [6] to avoid position effects (Fig 3A). We labeled these constructs GR-WT (fly wild type), GR-R1673P (Patient 1) and GR-R1664Q (Patients 2-5) corresponding to the human proteins/variants. These mutations are within the S4 transmembrane segment of domain IV that is nearly identical between flies and humans (Fig 3C).
The wild type P[acman] transgene rescues the lethality associated with Drosophila cac J and cac F (Fig 3D). However, GR-R1673P (Patient 1) mutation failed to rescue lethality, whereas GR-R1664Q (Patients 2-5) was able to rescue lethality partially (41% of expected viable progeny). This data suggest that the two mutations have functional consequences in vivo, and that the R1673P mutation seems to behave as a more severely impaired allele.

Electroretinograms reveals differential effects in CACNA1A alleles
Next we performed ERGs [6] in cac mutant clones rescued with either a wild type or a mutant P[acman] GR construct. ERG recordings reveal two key features: the 'on' and 'off' transients (red dotted circles, Fig 3E) typically reflect synaptic transmission between pre-and post-synaptic cells, whereas the amplitude of the depolarization (red bracket in Fig 3E) is a measure of the phototransduction activity. cac mutations typically affect the 'on' and 'off' transients but normal in size. B) Patient 1, a 3D FFE image at 22 months again showing a mid-sagittal view, with some increased prominence of the cerebellar folia suggesting early cerebellar atrophy C)Patient 1, T1 mid-sagittal view at 3.5 years showing progressive cerebellar atrophy D)Patient 1, a mid-sagittal T1 3D image at 8 years showing the neurodegenerative progression affecting the cerebellum E) Patient 3, a mid-sagittal T1 image at age 2 years, with normal cerebellum and thin corpus callosum posteriorly F) Patient 5, a mid-sagittal T1 image at age 18 months, normal.
https://doi.org/10.1371/journal.pgen.1006905.g001 have little or no effect on the amplitude, consistent with the role of cac in synaptic vesicle release [6]. To assess the function of the variants from the subjects, we generated homozygous mutant eye clones of cac J and cac F in young animals (3 days old) and compared the ERGs in flies that carry the wild type and mutant P[acman] GR constructs. Both cac alleles exhibit loss of synaptic transmission as evidenced by loss of the 'on' and 'off' transients (Fig 3E, S2A Fig).
Mutant flies carrying the wild type P[acman] cac transgene exhibited normal 'on' and 'off' transients ( Fig 3E, S2A Fig). Interestingly, GR-R1673P (Patient 1) dramatically increased the amplitude of the on and off transients, whereas GR-R1664Q (Patients 2-5) failed to rescue the synaptic transmission defect caused by the cac J and cac F mutations. These data suggest that the R1673P allele seen in Patient 1 is a gain-of-function mutation, and R1664Q found in Patients 2-5 is a loss-of-function allele based on the ERG defects observed in the photoreceptors in Drosophila. We also examined the 'on' and 'off' transients of flies with the P[acman] transgenes in a wild-type y w (cac +/cac+) background as well as heterozygous backgrounds (cac J /+ and cac F /+). Although there was a slight nominal increase in the 'on' transient in the R1673P animals, it was not statistically significant (S2B Fig).
As increased Ca 2+ influx typically causes excitotoxicity which may lead to the demise of neurons [6], we recorded the ERGs of 8 genotypes (cac F or cac J mutations rescued by GR-WT, GR-1673P, GR-1664Q, or no rescue) in 30-day-old flies. We observed a loss of depolarization amplitude in the R1673P (Patient 1) flies in the cac F (missense) mutant background (Fig 4A). In contrast, GR-R1664Q (Patients 2-5) did not lead to a significant decrease in amplitude. Interestingly, when the transgenes were tested in the cac J (nonsense) mutant background the reduction in amplitude for R1673P was less severe although still statistically significant (S2C Fig). Similar to those tested in cac F background, the R1664Q variant did not lead to a significant decrease in amplitude (S2C Fig). These data suggest that the increase in activity observed in young flies that carry the R1673P variant leads to age-dependent deterioration of the phototransduction pathway, a phenotype that has not been previously observed in cac loss-of-function mutations [6].

Ultrastructure of Drosophila photoreceptors with CACNA1A alleles
To assess if the R1673P allele may also affect the ultrastructure of the photoreceptors, we performed transmission electron microscopy (TEM). 30-day-old cac F mutant flies show a slight change in retinal morphology, but the photoreceptor cell bodies retain all seven rhabdomeres and normal overall structure compared to cac F rescued by the wild type cac containing P [acman] clone (Fig 4C versus 4B). Moreover, the cac F mutants rescued with GR-R1664Q (Patients 2-5) did not exhibit obvious morphological defects of photoreceptors at 30 days ( Fig  4D). In contrast, the cac F mutant photoreceptors rescued by GR-R1673P (Patient 1) show obvious features of photoreceptor neurodegeneration (Fig 4E). The rhabdomeres of the photoreceptors are severely disrupted (arrows), and the cytoplasms of these cells are filled with autophagic vesicles implying neurodegeneration. The severe neurodegenerative phenotype  observed here has never been seen in any cac alleles, including the null alleles, thus suggesting that R1673P may act via a (toxic) gain-of-function mechanism.
We also performed TEM at the level of the lamina, where the presynaptic photoreceptors make synaptic connections with post-synaptic neurons. These data show even more dramatic differences in phenotype between the cac F mutants rescued by wild type and mutant P[acman] transgenes (Fig 5). cac F rescued by the wild type cac P[acman] clone displayed normal morphology of six photoreceptor terminals (green areas in Fig 5A). Consistent with our previous findings [6], the cac F mutant exhibited aberrantly expanded terminals, accumulation of autophagic vacuoles (AVs) and some signs of synaptic degeneration (red area in Fig 5B). The P[acman] clone containing the R1664Q (Patients 2-5) variant partially rescued this terminal expansion phenotype (Fig 5C), whereas the cac F mutant rescued by GR-R1673P (Patient 1) shows smaller size of their terminals (Fig 5D, Quantification in Fig 5E), a phenotype which is likely due to the depletion of more synaptic vesicles with gain of channel function. In addition, we saw evidence of a dramatic synaptic degeneration in the GR-R1673P rescued flies (red outlined areas in Fig  5D) compared to the other genotypes. These data indicate that the R1673P mutation causes severe neurodegeneration in both photoreceptor cell bodies and terminals, likely due to gain of channel function that is toxic to neurons during aging.
Interestingly, we did not observe any severe neurodegeneration in cac J (nonsense) mutants rescued by GR-R1673P in both retinae and laminae (S3 and S4 Figs). Instead, we observed partial rescue of the terminal expansion phenotype, and notably autophagic vacuoles did not accumulate (S4D Fig). The latter suggests that lysosome-autophagosome fusion function is still present in cac with the R1673P mutation. The differences in the phenotypes observed in cac F and cac J mutants rescued by GR-R1673P further support that R1673P is a gain-of-function variant. In the cac F partial loss-of-function background, the R1673P variant can generate toxic gain-of-function effects that lead to severe neurodegeneration. In contrast, the cac J null allele expresses no endogenous cac and therefore alleviates the toxicity that arises from the R1673P variant. In summary, our experiments in Drosophila strongly suggest that the R1673P and R1664Q mutations are likely to be functional in human and likely to exert their effects through distinct mechanisms.

Discussion
We report five individuals with similar clinical presentations of ataxia, expressive speech delay, motor incoordination, and age of onset. The severe early-onset ataxias seen in these patients are similar to reports of severe early-onset ataxia associated with CACNA1A missense variants  circles). Quantifications of the on-transients and off-transients of ERG traces are on the right. Data are presented as mean ± SEM. p values were calculated using Student's t test. ***p < 0.001; NS, not significant. p-values were consistent with those on one way ANOVA. In 3-day-old cac F mutant clones carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) the electroretinogram has a normal pattern of synaptic activity (demonstrated by on-and off-transients) and depolarization (amplitude marker). In photoreceptors from cac F mutants there is a loss of "on" and "off." In the cac F mutants with the mutant transgenes (GR-R1673P, GR-R1664Q) there are distinct findings with R1673P producing larger "on" and "off" transients while the R1664Q produces a loss-offunction phenotype similar to the cac F mutants.
https://doi.org/10.1371/journal.pgen.1006905.g003 observed in the S4 transmembrane segment of domain III (e.g. p.I1342T, p.V1396M, and p. R1352Q) [27] or domain IV (R1664Q) [34]. Given these unique clinical features, it had been proposed that these represented loss-of-function mutations of the calcium channel [34]. These severe ataxia phenotypes were thought to represent the most severe end of the spectrum of EA2 rather than the gain-of-function mechanisms seen in hemiplegic migraines [29,34]. However, in our series, Patient 1, a girl with a de novo R1673P variant, also exhibited a progressive cerebellar neurodegenerative process of the most severe end of the CACNA1A clinical spectrum.
Our Drosophila studies indicate distinct functional consequences when comparing R1673P and R1664Q alleles. Initially we observed the R1673P allele failed to rescue lethality whereas R1664Q partially rescued, suggesting R1673P is a more severe allele. Importantly, the R1673P allele causes a neurodegenerative phenotype based on functional and morphological criteria which was not seen in either R1664Q or in loss-of-function alleles of cac. Despite the overall clinical similarity between the patients, the two alleles exhibit dramatic functional differences in Drosophila, a functional spectrum not observed previously in severe CACNA1A variants. In retrospect, we note that Patient 1, the only subject with the R1673P variant, had progressive neurodegeneration of the cerebellum, which was a distinguishing feature between her and the other four subjects. The molecular mechanism for the special arginine at position 1673 in regulating the Ca 2+ channel function remains unclear. Interestingly, the recently solved crystal structure of CaV1.1 suggests that both R1664 and R1673 are positively charged residues within the voltage sensor [43]. Since many disease-associated variants are found in positively charged residues in the S4 segment of domain IV of CACNA1A (Fig 2A'), to explore their functional differences will be an exciting future topic to eventually establish a mechanistic model for these key residues in Ca 2+ channel function.
Responsiveness to treatment and medication differs between reported cases of loss-of-function and gain-of-function CACNA1A alleles [11,18]. Patients with EA2 and loss-of-function alleles are often responsive to acetazolamide, while patients with gain-of-function alleles and FHM may respond but generally tend to be less responsive. One would predict that calcium channel blockers might be more effective for gain-of-function alleles. Whether patients with the more severe ataxias also differ clinically in their response to treatment remains to be tested. We note that, while Patient 5 had a strong positive response to acetazolamide, Patient 1 did not respond, consistent with this observation. Indeed as a result of our study Patient 1 was started on a calcium channel blocker. In conclusion, deciphering the functional impact of these severe CACNA1A alleles may provide insight into the pathogenic mechanisms and help direct therapeutic interventions.

Materials and methods
Human studies and whole exome sequencing  Clinically severe CACNA1A alleles affect synaptic function and neurodegeneration differentially National Human Genome Research Institute IRB, and in the Baylor-Hopkins Center for Mendelian Genomics protocol, H-29697, and "Evaluation of Sequence Variants" H-22769 approved by the IRB for Baylor College of Medicine). All families gave written informed consent for whole exome sequencing and publication. All sequencing studies were performed on genomic DNA from blood samples. Patient 1 obtained trio-based exome sequencing through BHCMG [31]. In brief, for patient 1 DNA samples were obtained and prepared into Illumina paired-end libraries and whole-exome capture with BCM-HGSC core design (52 Mb, Roche NimbleGen), and then sequencing on the Illumina HiSeq 2000 platform (Illumina). The produced data were aligned and mapped to the human reference genome (hg19) through the Mercury pipeline [44]. Single-nucleotide variants (SNVs) were called with the ATLAS (an integrative variant analysis pipeline optimized for variant discovery) variant calling method and annotated by the in-house Cassandra annotation pipeline that adapts ANNOVAR (Annotation of Genetic Variants) and additional inhouse tools [45][46][47]. De novo variants were calculated by an in-house developed pipeline (DNM-Finder) [31] for in silico subtraction of parental variants from the proband's variants in vcf files while accounting for the read number information extracted from BAM files. Bioinformatic tools predicted conservation and pathogenicity of candidate variants, and variants were compared against both an internal database and public databases such as the Exome Aggregation Consortium (ExAC) database.
Patients 2-5 had clinical proband exome sequencing at BGL [32,33] Fly genetics cac F and cac J mutants were isolated from a chemical mutagenesis screen as described previously [6,48]. The mapping and sequencing of the mutants was performed as described [49]. The P[acman] BAC construct that contains the full length cac genomic region was selected from a large P[acman] library that we previously described [49]. A Transgenic line from this BAC (CH321-60D21) was generated previously and named Dp(1;3)DC131 [49] which we refer to as GR-WT in this paper. Two point mutations (R1664Q or R1673P) were introduced into the CH321-60D21 BAC by recombineering using the modified DH10B strain SW102 and a galK positive/counter selection cassette [50]. The reagents for recombineering were obtained from Biological Resources Branch at National Cancer Institute (NCI) -Frederick. The microinjections to generate transgenic flies that contain wild type or mutant 77kb P[acman] clones were performed by Geneti-Vision, Houston, TX.
Lethality rescue experiments in Drosophila were done by crossing y w, cac F FRT19A/FM7c or y w, cac J FRT19A/FM7c with y w/Y; Dp(1;3)DC131(or-R1673P or -R1664Q) flies. The males that have no FM7c marker in the next generation were considered rescued flies. The numbers of rescued flies were also compared with FM7c males in the same progeny. synapse onto the laminar neurons. Clones carrying a wild type 80 kb P[acman] genomic rescue transgene (GR-WT) have a normal pattern of six Drosophila photoreceptors. B) cac F mutant photoreceptor clones with expansion and degenerative changes. C) cac F mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1664Q missense variant with reduced expansion. D) cac F mutant photoreceptor clones carrying an 80 kb P[acman] genomic rescue transgene with a missense change corresponding to the R1673P missense variant with dramatic neurodegeneration evidenced by loss of the normal ultrastructure, and apparent filling with disorganized electron dense material (red outlined areas) involving the synapsing photoreceptors. E) Quantification of capitate projections (CPs), active zones (AZs), mean terminal areas, autophagic vesicles per expanded terminal, and degenerative terminals per cartridge, respectively. Data are presented as means ± SEM. p values were calculated using Student's t test. ***p < 0.001; *p<0.05; NS, not significant. p-values were consistent with those on one way ANOVA for all groups. https://doi.org/10.1371/journal.pgen.1006905.g005

Generation of cac mutant eye clones in Drosophila
In order to circumvent the lethal phenotype, we generated mosaic clones in the Drosophila eyes for ERG and EM experiments. Virgin females of y w, cac F (or cac J ) FRT19A / FM7c; Dp (1;3)DC131-R1673P(or +) / CyO were crossed with cl(1) Ã FRT19A/ Y; ey-FLP males and we examined y w, cac F (or cac J ) FRT19A / cl(1) Ã FRT19A; ey-FLP/ Dp(1;3)DC131-R1673P(or +) progeny for ERG and EM defects. Homozygous mutant cells were marked by wand heterozygous cells were marked by w + . Homozygous wild-type cells were eliminated by the recessive cell lethal mutation (cl(1) Ã ) to give the mutant clones a growth advantage.

Electroretinogram (ERG) recording
ERG recordings were performed as previously described [51]. Briefly, adult flies were glued to a glass slide, a recording electrode was placed on the surface of the eye while a reference probe was inserted in the thorax. A fly eye was exposed to a flash of white light for 1 second. Responses were recorded and analyzed with AXON TM -pCLAMP8 software. Data were analyzed by two-tailed unpaired Student's t test. A p-value of <0.05 was considered statistically significant.

Transmission electron microscopy (TEM)
Laminae in adult flies were processed for TEM imaging as described [52]. Samples were processed using a Ted Pella Bio-Wave microwave oven with vacuum attachment. Adult fly heads were dissected at 25˚C in 4% paraformaldehyde, 2% glutaraldehyde, and 0.1 M sodium cacodylate (pH 7.2). Samples were subsequently fixed at 4˚C for 48 hours. 1% osmium tetroxide was used for secondary fixation and subsequently dehydrated in ethanol and propylene oxide, and then embedded in Embed-812 resin (Electron Microscopy Science, Hatfield, PA). 50 nm ultra-thin sections were obtained with a Leica UC7 microtome and collected on Formvar-coated copper grids (Electron Microscopy Science, Hatfield, PA). Specimens were stained with 1% uranyl acetate and 2.5% lead citrate and imaged using a JEOL JEM 1010 transmission electron microscope with an AMT XR-16 mid-mount 16 megapixel CCD camera.

Drosophila genetics
The genotypes of the fly strains used are as follows: