© 2020. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2020) 13, dmm042390. doi:10.1242/dmm.042390

RESEARCH ARTICLE Frameshift of YPEL3 alter the sensory circuit function in Drosophila Jung Hwan Kim1,*, Monika Singh1, Geng Pan2, Adrian Lopez1, Nicholas Zito1, Benjamin Bosse1 and Bing Ye2,*

ABSTRACT suggest that YPEL3 suppresses the epithelial-to-mesenchymal β A frameshift in Yippee-like (YPEL) 3 was recently found from transition in cell lines by increasing GSK3 expression a rare human disorder with peripheral neurological conditions (Zhang et al., 2016). Other studies have shown the role of YPEL including hypotonia and areflexia. The YPEL family is highly in development. The loss-of-function mutations of YPEL conserved from yeast to human, but its members’ functions are poorly orthologs in ascomycete fungus altered fungal conidiation and defined. Moreover, the pathogenicity of the human YPEL3 variant is appressoria development (Han et al., 2018). In zebrafish, a ypel3 completely unknown. We generated a Drosophila model of human morpholino-mediated targeting of altered brain structures YPEL3 variant and a genetic null allele of Drosophila homolog of (Blaker-Lee et al., 2012). YPEL3 YPEL3 (referred to as dYPEL3). Gene-trap analysis suggests that Recently, a mutation in human was found in a patient with dYPEL3 is predominantly expressed in subsets of neurons, including a rare disorder that manifests a number of neurological symptoms larval nociceptors. Analysis of chemical nociception induced by allyl- [the National Institutes of Health (NIH)-Undiagnosed Diseases isothiocyanate (AITC), a natural chemical stimulant, revealed Program]. The mutation was caused by duplication of a nucleotide YPEL3 reduced nociceptive responses in both dYPEL3 frameshift and null in a coding exon of , resulting in a frameshift and mutants. Subsequent circuit analysis showed reduced activation of consequently a premature stop codon. The clinical observation second-order neurons (SONs) in the pathway without affecting showed that the patient had normal cognition but manifested nociceptor activation upon AITC treatment. Although the gross peripheral symptoms, including areflexia and hypotonia. However, YPEL3 axonal and dendritic development of nociceptors was unaffected, whether the identified mutation is pathogenic in the nervous the synaptic contact between nociceptors and SONs was decreased system is unknown. Moreover, little is known about the functions of by the dYPEL3 mutations. Furthermore, expressing dYPEL3 in larval YPEL3 in the nervous system. Drosophila nociceptors rescued the behavioral deficit in dYPEL3 frameshift In the present study, we generated a model of the YPEL3 mutants, suggesting a presynaptic origin of the deficit. Together, human condition by creating the disease-relevant variant these findings suggest that the frameshift mutation results in YPEL3 using CRISPR/Cas9-mediated in-del mutations. Our gene-trap loss of function and may cause neurological conditions by weakening analysis suggests that subsets of neurons, including nociceptors, Drosophila YPEL3 dYPEL3 synaptic connections through presynaptic mechanisms. express the homolog of (referred to as ). Subsequent analysis revealed reduced nociceptive behavior in KEY WORDS: YPEL3, Pathogenicity, Rare mutation, Synaptic dYPEL3 mutants. Consistently, we found that dYPEL3 mutations connection impaired the activation of second-order neurons (SONs) in the nociceptive pathway and reduced the synaptic contact between INTRODUCTION nociceptors and these SONs. We further demonstrate that the YPEL3 belongs to the Yippee-like gene family, which is composed behavioral, circuit and cellular phenotypes in the dYPEL3 of a number of genes in eukaryotic species ranging from yeast to frameshift mutants are recapitulated in a genetically null allele of human (Hosono et al., 2004). Only a handful of studies have hinted dYPEL3, and that expressing wild-type dYPEL3 in nociceptors at the biological roles of YPEL3. YPEL3 was initially identified as a rescues the altered nociceptive behavior in the frameshift mutants. small unstable apoptotic because of its low protein stability These findings suggest that the identified human YPEL3 mutation is and the ability to induce when overexpressed in a myeloid pathogenic and affects neuronal synapses through a loss-of-function cell line (Baker, 2003). Subsequent studies implicated YPEL3 as a mechanism. tumor suppressor. YPEL3 expression correlates with activity (Kelley et al., 2010). Overexpression and knockdown analyses RESULTS Generation of a disease-relevant variant of YPEL3 in

1Department of Biology, University of Nevada, Reno, Reno, NV 89557, USA. 2Life Drosophila Sciences Institute and Department of Cell and Developmental Biology, University of Although the discovery of a YPEL3 variant in a patient underscores Michigan, Ann Arbor, MI 48109, USA. the importance of YPEL3 in human health, whether this variant *Authors for correspondence ( [email protected]; [email protected]) causes any deficits in the nervous system is unknown. There are five YPEL genes in human: YPEL1-YPEL5. YPEL1, YPEL2, YPEL3 J.H.K., 0000-0001-8548-4435; B.Y., 0000-0002-8828-4065 and YPEL4 are highly homologous to each other (up to 96% ∼ This is an Open Access article distributed under the terms of the Creative Commons Attribution identity at sequences), whereas YPEL5 has only 40% License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, to the other members (Hosono et al., 2004). We found distribution and reproduction in any medium provided that the original work is properly attributed. two YPEL homologs in Drosophila, Yippee and CG15309, using an Handling Editor: Steven J. Clapcote ortholog search (Hu et al., 2011). The predicted amino acid

Received 12 September 2019; Accepted 31 March 2020 sequences of CG15309 showed 88% similarity (81% identity) to Disease Models & Mechanisms

1 RESEARCH ARTICLE Disease Models & Mechanisms (2020) 13, dmm042390. doi:10.1242/dmm.042390 human YPEL3 (Fig. 1A), while that of Yippee showed 65% NIH-Undiagnosed Diseases Program). We thus focused our similarity (53% identity) (data not shown). Yippee appears to be an analysis on dYPEL3-positive neurons in the PNS (Fig. 2Bii,iii). ortholog of YPEL5 because it is more closely related to YPEL5 than The enhancer-trap analysis suggests that nociceptors express YPEL3, with 87% similarity and 73% identity to YPEL5 (data not dYPEL3. This finding was confirmed by co-immunostaining with shown). Therefore, we named CG15309 as dYPEL3. the nociceptor marker anti-Knot antibody (Hattori et al., 2007; The variant identified in the human patient introduces an extra Jinushi-Nakao et al., 2007) (Fig. 3A). The nociceptors detect nucleotide in the middle of the coding exon, which produces a various stimuli, including noxious heat, touch and chemicals, and frameshift and consequently results in the incorporation of the 37 activate the nociceptive pathway that leads to the nocifensive rolling ectopic amino acids followed by a premature stop codon (Fig. 1B). behavior (Hwang et al., 2007). Allyl-isothiocyanate (AITC), a To generate a Drosophila model of the human variant, we took natural chemical stimulant, has been proposed to cause larval advantage of the CRISPR/Cas9 technology to induce in-del nociceptive behavior through nociceptors (Kaneko et al., 2017; mutations (Port et al., 2014). The entire coding sequence of Xiang et al., 2010; Zhong et al., 2012). Since AITC also elicits dYPEL3 is in a single exon. We designed a guide RNA that targets nociceptive behavior in adult flies through gustatory sensory the middle of the coding exon (Fig. 1C, top) and successfully neurons (Soldano et al., 2016), we determined whether AITC- isolated two dYPEL3 frameshift mutants named dYPEL3T1-6 and induced nocifensive rolling required the larval nociceptors. dYPEL3T1-8 (Fig. 1C, middle). dYPEL3T1-6 has a two-nucleotide Optogenetic inhibition of larval nociceptors with Guillardia theta deletion at 121 nucleotides downstream of a start codon, which anion channel rhodopsin-1 (GtACR1) (Mohammad et al., 2017) generated a premature stop codon at 153 nucleotides downstream of dramatically reduced AITC-induced rolling (Fig. S3), a start codon, while dYPEL3T1-8 carries a four-nucleotide deletion at demonstrating that the AITC-induced rolling depends on the 118 nucleotides downstream of a start codon, and generated a nociceptors on the larval body wall. premature stop codon at 145 nucleotides downstream of a start We first determined whether the functions of nociceptors were codon. Similar to the human variant, the mutations introduced altered by the dYPEL3 mutations. We applied AITC to the wild-type additional amino acids followed by a premature stop codon (Fig. 1C, control, dYPEL3T1-6 and dYPEL3T1-8 and found a significant middle). The ectopic amino acids in dYPEL3T1-6 closely resemble reduction in nociceptive rolling behavior in the dYPEL3 mutants those of the human variant (Fig. 1C, bottom). (48% and 40% reduction, respectively, Fig. 3B). The extent of decrease in nociceptive rolling was not different between the two dYPEL3 is expressed in subsets of neurons mutant alleles of dYPEL3, which are almost identical except for the We did not find any gross developmental defects in dYPEL3T1-6 or sequences in the ectopic stretch of amino acids (Fig. 1C). This dYPEL3T1-8 flies. Homozygotes were viable and fertile, and showed suggests that the truncation of dYPEL3, but not the presence of the normal growth under standard culture condition (data not shown). ectopic amino acid sequences, is responsible for the observed This raises the possibility that dYPEL3 is expressed in a subset of phenotype. dYPEL3T1-8 represents a simpler version since it only cells in the body. Our efforts of generating antibodies against has incorporation of a few ectopic amino acids (Fig. 1C). Therefore, dYPEL3 failed in two independent trials, precluding the use of we focused our analysis on dYPEL3T1-8 for further analysis. immunostaining for identifying the cell types that express dYPEL3. How does dYPEL3 mutation affect the sensory function? We first We thus took advantage of a GAL4 enhancer-trap line, CG15309- looked into whether the dYPEL3 mutation affects the development GAL4 (dYPEL3-GAL4) (Gohl et al., 2011), to study the expression of nociceptors. We expressed mCD8::GFP specifically in pattern of dYPEL3 in flies. This line contains a GAL4 insertion in nociceptors in wild-type and dYPEL3T1-8 larvae using the the first intron of dYPEL3, which places the GAL4 under the control nociceptor-specific driver ppk-GAL4 (Grueber et al., 2007). The of the endogenous dYPEL3 and enhancers (Fig. 2A, top). dendritic arborization was assessed using Sholl analysis (Sholl, We expressed a membrane GFP reporter (mouse CD8::GFP or 1953) and by measuring total dendritic length. dYPEL3T1-8 mCD8::GFP) to visualize dYPEL3 expression pattern in Drosophila mutations did not alter the dendritic development (Fig. 4A,B). larvae. A small number of cells in the larval central nervous system Next, we tested whether the presynaptic terminals of nociceptors are (CNS), including the ventral nerve cord and brain, were labeled by defective in dYPEL3 mutants. To this end, a flip-out mosaic mCD8::GFP (Fig. 2A, bottom). These cells extended fine processes experiment was performed to label single nociceptive presynaptic that cover most of the neuropil area in the larval CNS, suggesting arbors (Yang et al., 2014). The total length of the presynaptic arbor that they are neurons. To identify the cell types that express of each nociceptor was indistinguishable between wild type and dYPEL3, dYPEL3-GAL4>mCD8::GFP samples were co- dYPEL3T1-8 (Fig. 4B), suggesting that dYPEL3T1-8 does not affect immunostained with the neuron marker anti-Elav and the glial the development of presynaptic arbors. marker anti-Repo (Fig. 2B). Approximately 85% of cells that were labeled with dYPEL3-GAL4 were positive for Elav, but none were The disease-relevant mutations of dYPEL3 reduce the positive for Repo (Fig. 2C). This result suggests that dYPEL3 is synaptic transmission from nociceptors to their predominantly expressed in neurons, but not in glia. Interestingly, postsynaptic neurons dYPEL3-GAL4 also labeled a subset of sensory neurons, including Next, we assessed the synaptic transmission from nociceptors to the class IV dendritic arborization (da) neurons (nociceptors), class their postsynaptic neuron Basin-4, a key SON in the nociceptive III da neurons and chordotonal neurons (both mechanosensors), but pathway (Ohyama et al., 2015). The activation of Basin-4 elicits not the class I da neurons (proprioceptors) (Fig. 2Bii,iii; Fig. S1). nociceptive behavior even in the absence of nociceptor activation, dYPEL3 was not expressed in muscles or epidermal cells (Fig. S2). while silencing these neurons suppresses nociceptive behavior (Ohyama et al., 2015). The genetically encoded calcium indicator The disease-relevant mutations of dYPEL3 reduce GCaMP6f was selectively expressed in Basin-4 for recording nociceptive behavioral responses intracellular calcium, a proxy of neuronal activity (Chen et al., 2013) The human patient shows symptoms mainly in the peripheral (Fig. 5A). Larvae were dissected in insect saline as a fillet nervous system (PNS), including areflexia and hypotonia (the preparation with intact PNS and CNS (Kaneko et al., 2017) and Disease Models & Mechanisms

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Fig. 1. The generation of a Drosophila model of YPEL3 frameshift mutation. (A) CG15309 is the Drosophila homolog of human YPEL3. Sequence alignment between human YPEL3 (YPEL3) and Drosophila CG15309. Shaded in pink are the identical amino acid sequences. (B) Duplication of a cytosine nucleotide in YPEL3 gene from a patient (top). A predicted molecular lesion in human YPEL3 (bottom) introduces an ectopic amino acid sequence (shaded in green). The preserved region is shaded in pink. (C) CRISPR-Cas9 mediated in-del mutation in CG15309/dYPEL3. A guide RNA is designed targeting the middle of the coding exon (top). The isolated dYPEL3 in-del mutants (middle). Sequence alignment between wild type (wt), dYPEL3T1-6 and dYPEL3T1-8. The introduced ectopic amino acid sequences following a premature stop codon are shaded in green. The sequence alignment of the introduced ectopic amino acid sequences from the human YPEL3 frameshift mutants and dYPEL3T1-6 (bottom). The identical amino acid sequences are shaded in pink. ORF, open reading frame. treated with AITC to stimulate the nociceptors. AITC elicited robust dYPEL3T1-8 mutants, compared to wild-type control (Fig. 5A, 43% GCaMP signals that persisted over several minutes in both decrease). By contrast, GCaMP measurement in nociceptor axon nociceptors and Basin-4 neurons (Fig. 5A,B). We found that the terminals showed that dYPEL3T1-8 did not change AITC-induced

GCaMP signals in Basin-4 neurons were significantly decreased in activation of nociceptors (Fig. 5B). Disease Models & Mechanisms

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Fig. 3. dYPEL3 frameshift mutations reduce nociceptive behavior. (A) Nociceptive/class IV da neurons are positive for dYPEL3. A nuclear GFP (GFP-nls, green) was expressed under dYPEL3-GAL4 following immunostaining with anti-Knot antibody (blue). Anti-HRP antibody was used to label all PNS neurons (magenta). Scale bar: 10 μm. (B) The AITC-induced nociceptive behavior was measured in a wild-type control (wt) and dYPEL3 frameshift mutants (dYPEL3T1-8 and dYPEL3T1-6). The number of larvae that exhibited complete rolling behavior was scored and expressed as a percentage (n=252 for each genotype). The Chi-squared test was performed between the groups. NS, non-significant; ****P<0.0001.

the synaptic contact between the presynaptic terminals of nociceptors and the dendrites of Basin-4 neurons. The GRASP technique utilizes two separate fragments of GFP molecule – split-GFP1-10 (spGFP1- 10) and split-GFP11 (spGFP11), which can be detected by a specific anti-GFP antibody only when the two fragments are in close proximity to reconstitute a complete GFP. In syb-GRASP, spGFP1- Fig. 2. dYPEL3 is a neuronal gene. (A) The expression pattern of dYPEL3 in the CNS. The InSITE gene trap line for dYPEL3 was used (CG15309-GAL4/ 10 is fused to the synaptic vesicle protein Synaptobrevin and dYPEL3-GAL4). GAL4 transcription factor is inserted in the first intron. The expressed in the presynaptic neurons, whereas spGFP11 is fused to a introduction of UAS-mCD8::GFP demonstrates the endogenous expression general membrane tag and expressed in postsynaptic neurons. Two pattern of dYPEL3. Note that the CG15309-GAL-positive cells elaborate fine independent binary gene expression systems, GAL4-UAS and LexA- processes throughout the CNS. Scale bar: 50 µm. (B) mCD8::GFP (magenta) LexAop, were used to drive the expression of spGFP1-10 and was expressed under dYPEL3-GAL4 following immunostaining with anti-Elav spGFP11 in different cell types (del Valle Rodríguez et al., 2012). (neuronal, green) and anti-Repo (glial, blue) antibodies. (i) The CNS. Cell Synaptic vesicle exocytosis from presynaptic terminals exposes bodies in 1 and 2 are shown magnified on the top right. (ii,iii) Chordotonal neurons (ii, arrows) and a class III da neuron (iii) in the PNS. iii also shows a spGFP1-10 onto the presynaptic cleft, where it reconstitutes the class IV da neuron (nociceptor) that is positive for dYPEL3 (arrow). Scale bar: functional GFP molecule by associating with postsynaptic spGFP11 10 µm. (C) Quantitation of the Elav-positive and Repo-positive cells that are molecules. This technique has been used widely to visualize labeled with CG15309-GAL4. The majority of dYPEL3-postive cells were Elav synaptic contact between two identified neuron types. positive, but none were positive for Repo. The spGFP1-10 and spGFP11 were specifically expressed in nociceptors and Basin-4 neurons, respectively (Fig. 6A, left). The The disease-relevant mutations of dYPEL3 reduce the resulting GRASP signal was measured in each segmental neuropil, synaptic contact between nociceptors and their and normalized by the spGFP1-10 intensity in wild type and in postsynaptic neurons dYPEL3T1-8 (Fig. 6A, right). We detected a mild, but significant, How do the dYPEL3 mutations reduce the nociceptor-to-Basin-4 decrease (23%) in the GRASP signals in dYPEL3T1-8, compared to synaptic transmission? To address this, we employed a synaptic- those in wild-type control (Fig. 6B). This suggests that the synaptic contact-specific GFP reconstitution across synaptic partners (GRASP) contact between nociceptors and its synaptic target Basin-4 is T1-8 technique, termed syb-GRASP (Macpherson et al., 2015), to assess compromised in dYPEL3 . Disease Models & Mechanisms

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Fig. 4. The development of nociceptors is not altered by dYPEL3 frameshift mutations. (A) mCD8::GFP was specifically expressed in nociceptors using ppk-GAL4 in wild-type control (wt) and dYPEL3 frameshift mutants (dYPEL3T1-8). Total length of dendrites was measured (n=6 for each genotype). Unpaired Student’s t-test with Welch’s correction was performed. Scale bar: 50 µm. (B) Sholl analysis was performed with 20-µm radius increment from the dendritic tracing. Number of total crossings within 300 µm from the cell center was measured. Two-way ANOVA for Sholl analysis and unpaired Student’s t-test with Welch’s correction for total dendritic crossings were performed. (C) The axon terminals of single nociceptors from wild type and dYPEL3T1-8 mutants were visualized using the flip-out technique. The total length of axon terminals was measured (n=12 for wt, n=14 for dYPEL3T1-8). Scale bar: 10 µm. Unpaired Student’s t-test with Welch’s correction was performed. Data are presented as mean±s.e.m. All statistical analysis was two-tailed. NS, non-significant.

The disease-relevant frameshift mutant of dYPEL3 is a rescued defective rolling behavior induced by AITC (Fig. 8), loss-of-function allele whereas expressing dYPEL3 in wild type had no effect. The mutations in the patient and in our Drosophila model introduce Taken together, these results strongly support a model that the premature stop codons, which may induce the nonsense-mediated frameshift in human YPEL3 causes YPEL3 loss of function, and that decay (Chang et al., 2007), resulting in YPEL3 loss of function. YPEL3 acts in presynaptic neurons to positively regulate synaptic However, the frameshift mutation in YPEL3 may escape from contact. nonsense-mediated decay because the premature stop codons are in the last coding exons (both in human and Drosophila), which may DISCUSSION lead to the production of a truncated version of YPEL3 . To The biological functions of the YPEL gene family, including discern these possibilities, we generated a genetically null allele of YPEL3, are poorly understood. Moreover, whether the identified dYPEL3 (dYPEL3KO) by removing the entire dYPEL3 coding region YPEL3 frameshift mutation is pathogenic is unknown. Drosophila using CRISPR/Cas9 (Fig. 7A). We found that dYPEL3KO larvae provides a powerful tool for analyzing disease-relevant human gene recapitulated all the deficits found in dYPEL3 frameshift mutants to mutations (Bellen et al., 2019). In this study, we report a Drosophila the similar extent. These include AITC-induced rolling behavior model of human YPEL3 mutation and demonstrate that the disease- (40% decrease), AITC-induced Basin-4 activation (47% decrease), relevant YPEL3 frameshift mutations are pathogenic in the nervous and synaptic contact between nociceptors and Basin-4 (24% system. decrease) (Fig. 7B-D). These results strongly suggest that the The YPEL gene family is highly conserved across disease-relevant mutation of dYPEL3, dYPEL3T1-8, is a loss-of- ranging from yeast to human. Likewise, our homology analysis function allele. indicated a strikingly high homology in gene sequences between If dYPEL3 frameshift mutation is loss of function, the defects in human and Drosophila YPEL3 (80% identity, Fig. 1B). these mutants may be rescued by the expression of wild-type Interestingly, it appears that the extends even dYPEL3. The nociceptors, but not Basin-4 neurons, express dYPEL3 to the nucleotide level since the analogous frameshift mutation gave (Fig. 3A; Fig. S4). Thus, we expressed dYPEL3 specifically in rise to the generation of similar amino acid sequences in the ectopic nociceptors using ppk-GAL4 (Grueber et al., 2007) in wild-type and sequences in dYPEL3T1-6 (Fig. 1C). Given such high sequence dYPEL3T1-8 larvae and tested AITC-induced nociceptive behavior. homology, we envision that the functions of human YPEL3 and T1-8 We found that expressing dYPEL3 in dYPEL3 completely Drosophila YPEL3 are also conserved. The YPEL family can be Disease Models & Mechanisms

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Fig. 6. dYPEL3 frameshift mutations reduce the synaptic contact between nociceptors and Basin-4 neurons. (A) The syb-GRASP technique was used to report the synaptic contact between nociceptors and Basin-4. The spGFP1- 10 (red cylinders) and spGFP11 (blue sectors) were expressed in nociceptors and Basin-4, respectively (left). The resulting GRASP signal was visualized by anti-GRASP antibody (green), and the spGFP1-10 that is expressed in nociceptor axon terminals was used as a normalization control (magenta) (right). Scale bar: 10 μm. (B) The GRASP intensity from each neuropil was normalized by spGFP1-10 intensity and presented as mean± s.e.m. (left), as well as in a violin plot to show distribution (right) (n=36 for wt, n=34 for dYPEL3T1-8). Mann–Whitney test. All statistical analysis was two- tailed. **P<0.01.

subdivided into two categories. Human YPEL1, YPEL2, YPEL3 and YPEL4 belong to one with high homology with each other, while YPLE5 constitutes a distinct family (Hosono et al., 2004). In Drosophila, there is only a single homolog of human YPEL1- YELP4, CG15309 (Fig. 1B). Because the tissue expression patterns of YPEL genes are complex in human and mice (Hosono et al., 2004), the single YPEL gene makes Drosophila advantageous as a model for studying YPEL3-induced pathogenesis. In human and mice, YPEL3 is ubiquitously expressed, as based on results from RT-PCR experiments (Hosono et al., 2004). Northern blot analysis of murine tissues shows relative enrichment Fig. 5. dYPEL3 frameshift mutations reduce the synaptic transmission of YPEL3 in brain and liver tissue (Baker, 2003). Our results based from nociceptors to Basin-4 neurons. (A) Basin-4 activation upon AITC on a gene-trap Drosophila line indicates that dYPEL3 is expressed treatment was reduced by dYPEL3T1-8. GCaMP6f was expressed in Basin-4 in subsets of neurons, but not in glia (Fig. 2B,C). The human patient neurons. Nociceptors were activated with 10 mM AITC (top left). Ca2+ increase exhibited multiple neurological symptoms in the PNS, but had in Basin-4 was measured by GCaMP fluorescence and the tracing over time is normal cognition (the NIH-Undiagnosed Diseases Program). shown (n=40 for wt, n=47 for dYPEL3T1-8) (top right). The cumulative GCaMP activation from single Basin-4 neurons was measured and presented as mean± Interestingly, dYPEL3-GAL4 was selectively expressed in s.e.m. (bottom left), as well as in a violin plot to show distribution (bottom right). nociceptors and mechanosensors in the PNS (Fig. 3A; Fig. S2). Mann–Whitney test. (B) Nociceptor activation was not altered by dYPEL3 Furthermore, YPEL3 frameshift mutations reduced nociceptive mutations. GCaMP6f was expressed in nociceptors using ppk-GAL4. behavior (Fig. 3B). These results suggest that at least some of the Nociceptors were activated with 10 mM AITC (top left). Ca2+ increase in the axon neurological symptoms in the human patient originate from neurons terminals of nociceptors was measured by GCaMP fluorescence and the tracing that express YPEL3. n over time is shown ( =13 for each genotype) (top right). The cumulative GCaMP How does the YPEL3 frameshift mutation cause sensory deficits? activation from the nociceptor axon terminals was measured and presented as mean±s.e.m. (bottom left), as well as in a violin plot to show distribution (bottom The gross neuronal development of nociceptors was not altered by right). Mann–Whitney test. All statistical analysis was two-tailed. NS, non- the dYPEL3 mutations (Fig. 4). Calcium-imaging experiments significant; ****P<0.0001. showed that activation of nociceptors by AITC was not altered in Disease Models & Mechanisms

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Fig. 7. dYPEL3 knockout mutants recapitulate the phenotypes in the dYPEL3 frameshift mutants. (A) The generation of dYPEL3 knockout (dYPEL3KO) flies. Top: the entire dYPEL3 ORF was deleted using CRISPR/Cas9-mediated homology-directed recombination. Bottom: agarose gel image of PCR-based genotyping. Note that dYPEL3KO showed the PCR amplifications specific for DsRed-cassette, but not the ones from wild type. (B) dYPEL3 knockout reduces AITC-induced behavioral responses. AITC-induced nociceptive behavior was measured in a wild-type control (wt) and dYPEL3 knockout mutants (dYPEL3KO). The number of larvae that exhibited complete rolling behavior was scored and expressed as a percentage (n=252 for wt, n=203 for dYPEL3KO). The Chi-squared test was performed between the groups. (C) dYPEL3 knockout caused a reduction in Basin-4 activation upon AITC. GCaMP6f was expressed in Basin-4 neurons. Nociceptors were activated with 10 mM AITC. Ca2+ increase in Basin-4 was measured by GCaMP fluorescence and the GCaMP trace over time is shown (n=47 for wt, n=46 for dYPEL3KO) (left). The cumulative GCaMP activation from single Basin-4 neurons was measured and presented as mean±s.e.m. (middle), as well as in a violin plot to show distribution (right). Mann–Whitney test. (D) dYPEL3 knockout causes a reduction in syb-GRASP signals between nociceptors and Basin-4. Synaptobrevin-spGFP1-10 and spGFP11 were expressed in nociceptors and Basin-4, respectively. The resulting GRASP signal was visualized by anti-GFP antibody that only recognizes the reconstituted GFP (anti-GRASP) (green), and the spGFP1-10 expressed in nociceptor axon terminals was used as a normalization control (magenta) (left). Scale bar: 10 μm. The GRASP intensity from each neuropil was normalized by spGFP1-10 intensity and presented as mean±s.e.m. (middle), as well as in a violin plot to show distribution (right) (n=32 for wt, n=80 for dYPEL3T1-8). Mann–Whitney test. ***P<0.001 and ****P<0.0001. dYPEL3T1-8 mutants (Fig. 5B). Rather, dYPEL3T1-8 reduced Basin- nociceptive behavior (Ohyama et al., 2015), the reduced synaptic 4 responses to nociceptor stimulation (Fig. 5A). This suggests that transmission from nociceptors to Basin-4 is likely responsible for the neurotransmission from nociceptors to their postsynaptic the reduction in nociceptive behavior in dYPEL3 mutants. It is neurons is reduced by the dYPEL3 frameshift mutation. This intriguing that the human patient has peripheral symptoms of conclusion is corroborated by the finding that the syb-GRASP hypotonia and areflexia; both may arise from reduced synaptic signal between nociceptors and Basin-4 was reduced (Fig. 6). Since transmission. the syb-GRASP technique requires synaptic release (Macpherson We observed that AITC-elicited GCaMP signals persisted over et al., 2015), it is possible that the decrease in syb-GRASP signals a few minutes in both nociceptors and Basin-4 neurons (Figs 5A,B reflects reduced activity or synaptic release in nociceptors in the and 7C). Since AITC does not induce continuous larva rolling mutants. Alternatively, the reduction in syb-GRASP signals might over such a long period, this implies the presence of an acute be due to a reduced number of synapses in the mutants. Additional adaptation to AITC stimulation in the nociceptive circuit. It is techniques are needed to discern these possibilities. Nevertheless, possible that the ex vivo GCaMP measurement does not fully the results from calcium imaging and syb-GRASP experiments recapitulate neural activity in vivo. Nevertheless, our results consistently show reduced synaptic transmission from nociceptors indicate that the dYPEL3 mutations significantly reduce calcium to the SON Basin-4. Because Basin-4 activation is central to increase in Basin-4 neurons. Disease Models & Mechanisms

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maintenance. Future studies that identify the molecular mechanisms underlying the function of YPEL3 will provide insights into therapeutic treatments of disorders caused by YPEL3 mutations.

MATERIALS AND METHODS strains Drosophila strains were kept under standard condition at 25°C in a humidified chamber. The following strains were used: w1118 (3605), ppk- GAL4 (Grueber et al., 2007), ppk-LexA (Gou et al., 2014), UAS-syb:: spGFP1-10 (Macpherson et al., 2015), LexAop-CD4::spGFP11 (Macpherson et al., 2015), UAS-FRT-rCD2-stop-FRT-CD8::GFP (Wong et al., 2002), hs-FLP (Nern et al., 2011) (55814), UAS-CD4-GFP (35836), UAS-GCaMP6f (Mutlu et al., 2012) (42747), LexAop-GCaMP6f (Mutlu et al., 2012) (44277), CG15309-GAL4 (Gohl et al., 2011) (62791), nos- Cas9 (Port et al., 2014) (54591), GMR57F07-GAL4 (Jenett et al., 2012) (46389), GMR57F07-lexA (Pfeiffer et al., 2010) (54899), UAS-GtACR1 (Mohammad et al., 2017), NompC-lexA (Shearin et al., 2013) (52241) and a Cre-recombinase expressing fly line (1501, RRID:BDSC_1501). The numbers in parentheses indicate the stock numbers from the Bloomington Drosophila Stock Center.

The generation of dYPEL3 mutants dYPEL3 Fig. 8. The nociceptor-specific expression of dYPEL3 in dYPEL3 The CRISPR/CAS9-mediated in-del mutation was used to generate frameshift mutant rescues AITC-mediated larva rolling behavior. frameshift mutant flies. A guide RNA construct was generated in pCFD: AITC-induced nociceptive behavior was measured. The nociceptor-specific U6:3 (Port et al., 2014) with guide RNA sequences that target the middle of expression of dYPEL3 was achieved using ppk-GAL. The number of larvae the dYPEL3 coding exon. The standard transformation procedure was that exhibited complete rolling behavior was scored and expressed as a performed to generate a transgenic line. The transformants were crossed percentage (n=118 for wt, ppk>; n=115 for wt, ppk>dYPEL3; n=163 for with nos-Cas9 (Port et al., 2014) flies to induce in-del mutations in germ dYPEL3T1-8, ppk>; n=103 for dYPEL3T1-8, ppk>dYPEL3). The Chi-squared cells. The resulting progeny were screened for the desired mutations by the test was performed between the groups. NS, non-significant; *P<0.05. genomic PCR of CG15309 following the Sanger sequencing. The genetically null dYPEL3 allele (dYPEL3KO) was generated using How does the YPEL3 frameshift mutation affect YPEL3 gene CRISPR/Cas9-mediated homology directed recombination (HDR). The function? Our results suggest that YPEL3 frameshift mutations cause HDR donor construct was built using pBluescript as a backbone, which 3XP3:RFP loss of function. The behavioral, circuit and synaptic phenotypes were includes (for expressing DsRed in eyes) that is flanked by loxP dYPEL3 dYPEL3T1-8 sequences (Lin and Potter, 2016) and two homology arms (∼700 bp and almost identical between frameshift ( )and ∼ dYPEL3KO 1 kb for right and left arms, respectively). The homology arms were knockout ( ) mutants (Figs 3, 5, 6 and 7). Since w1118 dYPEL3 amplified from flies. Two guide RNA constructs that target near the nociceptors, but not Basin-4, express (Fig. 3A; Fig. S4), start and the end of dYPEL3 open reading frame (ORF) were cloned in dYPEL3 mutations likely affect presynaptic functions. Consistently, T1-8 pCFD:U6:3 (Port et al., 2014). The two guide RNA constructs and the HDR the behavioral phenotype in dYPEL3 was completely rescued by donor construct were co-injected into w1118, nos-Cas9 fly embryos. Flies expressing wild-type dYPEL3 in nociceptors (Fig. 8). Taken were screened for eye expression of DsRed, and successful integration of the together, these findings suggest that YPEL3 functions in donor construct was confirmed by a PCR-based genotyping. The eye- presynaptic neurons to regulate synaptic transmission, and that specific DsRed cassette was removed by crossing the flies carrying the frameshift mutations in YPEL3 result in loss of YPEL3. donor construct and those expressing Cre recombinase. Generation of UAS- dYPEL3 dYPEL3 The molecular function of YPEL3 is unclear. It contains was done using pUASTattB plasmid and ORF that was w1118 predicted zinc-finger motifs (Hosono et al., 2004). The zinc- amplified from genomic DNA. Standard methodology was used to generate transformants (Bischof et al., 2007). finger motifs in a YPEL domain of the yeast protein Mis18 is important for the folding of the YPEL domain, which mediates the AITC-induced nociceptive behavior centromeric localization of Mis18 (Subramanian et al., 2016). The ∼ AITC (Sigma-Aldrich) was prepared in DMSO, dissolved in water to a final YPEL domain in Mis18 has 20% sequence similarity to YPEL 25 mM concentration, and incubated on a rocker for 3 days before use. Fly proteins. Since zinc-finger motifs are common in regulators of gene embryos were grown for 5 days in a 12 h light/dark cycle at 25°C in a expression, we suspect that the frameshift mutation of YPEL3 may humidified incubator. The third-instar larvae were moved to room change gene expression. Indeed, overexpression of YPEL3 temperature for 1 h, gently scooped out of food, washed in tap water and increased the expression of GSK3β to suppress the epithelial- placed on a grape-agar 24-well plate that had been covered with 300 µl mesenchymal transition (Zhang et al., 2016). It is interesting to note AITC solution (25 mM). Their behavior was recorded with a digital camera that GSK3β has been implicated in synaptogenesis (Cuesto et al., for 2 min and the number of larvae showing complete rolling behavior (minimum 360° rolling) was manually analyzed (Honjo et al., 2012). The 2015). Thus, it will be important to determine whether YPEL3 w1118 dYPEL3 regulates the expression of genes involved in synapse formation and experiments were paired for the wild-type control ( ) and YPEL3 homozygous mutant larvae. Experiments were repeated three times on maintenance and investigate how frameshift mutations different days with different AITC preparations. All three trials were affect this process. combined for statistical analysis. Overall, we generated a Drosophila model of the human YPEL3 YPEL3 frameshift mutation and found that the variant leads to Calcium imaging deficits in synaptic transmission. We further demonstrate that the Live calcium imaging was performed using GCaMP6f (Mutlu et al., 2012). frameshift mutation causes loss of YPEL3 function. In addition, this Briefly, wandering third-instar larvae – wild-type control males or T1-8 study establishes YPEL3 as a regulator of synaptogenesis or dYPEL3 hemizygotes – were dissected in a modified hemolymph-like Disease Models & Mechanisms

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3 (HL3) saline (Stewart et al., 1994) (70 mM NaCl, 5 mM KCl, 0.5 mM immunostaining and imaging. The total presynaptic arbor length was CaCl2, 20 mM MgCl2, 5 mM trehalose, 115 mM sucrose and 5 mM manually measured using ImageJ software. Branches shorter than 5 µm HEPES, pH 7.2). Glutamate (10 mM) was added to the HL3 solution to were excluded from the analysis. prevent muscle contractions and sensory feedback. The GCaMP signal was recorded in the entire volume of nociceptor axon terminals or Basin-4 cell Experimental design and statistical analysis bodies. Live imaging was performed with a Leica SP5 confocal system or a All statistical analysis was performed two-tailed using Prism version 7.04 custom-built spinning disk microscope equipped with an extra-long- (GraphPad Software). The Chi-square with Fisher’s exact test was used for working distance 25× water objective with 2-µm step sizes. The nociceptive rolling behavior. The Mann–Whitney test was used for calcium membrane tdTomato proteins were expressed along with GCaMP6f and imaging (GCaMP) and GRASP experiments. Unpaired Student’s test was used as an internal normalization control for both lateral and focus drifting. used for presynaptic arbor size and dendritic development analysis. Two- The basal GCaMP signal was recorded for a duration of 30 s to generate way ANOVA was used for Sholl analysis. P<0.05 was considered F baseline fluorescence ( 0), and then the samples were treated with AITC statistically significant. (10 mM) in HL3 while being continuously recorded for an additional 150 s. The 3D time-lapse images were collapsed to 2D time-lapse images by using Acknowledgements the maximum Z-projection in ImageJ software (NIH). The region of interest We thank Heewon Lee and Lily Lou for technical assistance, Dr Adam Claridge- was selected in the axonal projection of nociceptors or in the cell bodies of Chang for sharing the UAS-GtACR1 transgenic flies and Dr Cynthia Tifft for helpful Basin-4. The ImageJ Time Series Analyzer plugin was used to quantify the discussions. fluorescence intensity of GCaMP6f. The cumulative GCaMP was calculated from the GCaMP tracing from AITC treatment (t=30 s) to the end of Competing interests recording (t=180 s). The authors declare no competing or financial interests.

Author contributions Immunostaining Conceptualization: J.H.K., B.Y.; Methodology: J.H.K., N.Z.; Validation: J.H.K., M.S., Immunostaining was performed essentially as previously reported (Kim G.P., A.L., N.Z.; Formal analysis: J.H.K., M.S., G.P., A.L., N.Z., B.B.; Investigation: et al., 2013). The primary antibodies used were as follows: chicken anti-GFP J.H.K., M.S., G.P., A.L., N.Z., B.B.; Resources: J.H.K.; Data curation: J.H.K.; Writing (AB_2307313, Aves Laboratories; 1:2500), rabbit anti-RFP (600-401-379- - original draft: J.H.K.; Writing - review & editing: J.H.K., M.S., B.Y.; Visualization: RTU, Rockland Immunochemicals; 1:5000), rat anti-Elav (9F8A9, J.H.K.; Supervision: J.H.K., B.Y.; Project administration: J.H.K.; Funding acquisition: Developmental Studies Hybridoma Bank; 1:100) and mouse anti-Repo J.H.K., B.Y. (8D12, Developmental Studies Hybridoma Bank; 1:5). The secondary antibodies were from Jackson ImmunoResearch and used at 1:500 dilution: Funding Cy2- or Cy5-conjugated goat anti-chicken, Cy2- or Cy5-conjugated goat Research reported in this study used the Cellular and Molecular Imaging Core facility anti-mouse, Cy5-conjugated goat anti-rabbit and Cy3-conjugated goat anti- at the University of Nevada Reno, which was supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under grant rat. Confocal imaging was performed with a Leica SP8 confocal system or a number P20 GM103650, and was supported by the Nevada INBRE (P20 custom-built spinning disk confocal microscope equipped with a 63× oil- GM103440 to J.H.K.) and NIH (R21GM114529 and R01NS104299 to B.Y.). immersion objective with 0.3-µm step size. The resulting 3D images were projected into 2D images using a maximum projection method. Supplementary information In order to report the relative synaptic contact between the nociceptors Supplementary information available online at and their postsynaptic partners, syb-GRASP was performed in the male http://dmm.biologists.org/lookup/doi/10.1242/dmm.042390.supplemental larvae from a wild-type control (w1118), dYPEL3T1-8 or dYPEL3KO hemizygotes. Syb::split-GFP1-10 (Macpherson et al., 2015) was References expressed in nociceptors. CD4::split-GFP11 (Macpherson et al., 2015) Baker, S. J. (2003). Small unstable apoptotic protein, an apoptosis-associated was expressed in Basin-4 neurons. The polyclonal chicken anti-GFP protein, suppresses proliferation of myeloid cells. Cancer Res. 63, 705-712. antibody (Aves Laboratories) recognizes the split-GFP1-10 and the Bellen, H. J., Wangler, M. F. and Yamamoto, S. (2019). The fruit fly at the interface of diagnosis and pathogenic mechanisms of rare and common human diseases. reconstituted GFP protein, while the mouse anti-GFP antibody (G6539, Hum. Mol. Genet. 28, R207-R214. doi:10.1093/hmg/ddz135 Sigma-Aldrich) recognizes only the reconstituted GFP. Therefore, the Bischof, J., Maeda, R. K., Hediger, M., Karch, F. and Basler, K. (2007). An mouse anti-GFP antibody was used to measure the GRASP signal (anti- optimized transgenesis system for Drosophila using germ-line-specific C31 GRASP; 1:100) and the polyclonal chicken anti-GFP antibody was used as integrases. Proc. Natl. Acad. Sci. USA 104, 3312-3317. doi:10.1073/pnas. an internal control for normalizing the GRASP signal. The fluorescence 0611511104 Blaker-Lee, A., Gupta, S., McCammon, J. M., De Rienzo, G. and Sive, H. (2012). images were acquired to minimum signal saturation for quantitation. The Zebrafish homologs of genes within 16p11.2, a genomic region associated with mean fluorescence intensities of anti-GRASP and anti-split-GFP1-10 from brain disorders, are active during brain development, and include two deletion each hemi-neuropil segment (segments 4, 5 and 6) were measured from the dosage sensor genes. Dis. Model. Mech. 5, 834-851. doi:10.1242/dmm.009944 confocal images. Chang, Y.-F., Imam, J. S. and Wilkinson, M. F. (2007). The nonsense-mediated decay RNA surveillance pathway. Annu. Rev. Biochem. 76, 51-74. doi:10.1146/ annurev.biochem.76.050106.093909 Assessment of dendrite development in nociceptors Chen, T.-W., Wardill, T. J., Sun, Y., Pulver, S. R., Renninger, S. L., Baohan, A., The membrane GFP, mCD8::GFP, was specifically expressed in nociceptors Schreiter, E. R., Kerr, R. A., Orger, M. B., Jayaraman, V. et al. (2013). using ppk-GAL4 in a wild-type control (wt) and dYPEL3 frameshift mutants Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, (dYPEL3T1-8). Total length of dendrites was measured from the male larvae 295-300. doi:10.1038/nature12354 of wt and dYPEL3T1-8 using the Simple neurite tracer plugin (Longair et al., Cuesto, G., Jordán-Álvarez, S., Enriquez-Barreto, L., Ferrús, A., Morales, M. β 2011) in ImageJ software. Sholl analysis was conducted using the Sholl and Acebes, Á. (2015). GSK3 inhibition promotes synaptogenesis in drosophila and mammalian neurons. PLoS ONE 10, e0118475. doi:10.1371/journal.pone. analysis plugin in ImageJ software (Ferreira et al., 2014). 0118475 del Valle Rodrıguez,́ A., Didiano, D. and Desplan, C. (2012). Power tools for gene Analysis of presynaptic arbors of single nociceptors expression and clonal analysis in Drosophila. Nat. Methods 9, 47-55. doi:10.1038/ The flip-out (Wong et al., 2002) experiment was performed to visualize the nmeth.1800 terminal axon arbors of single nociceptors. A flip-out cassette (FRT-rCD2- Ferreira, T. A., Blackman, A. V., Oyrer, J., Jayabal, S., Chung, A. J., Watt, A. J., stop-FRT-CD8::GFP) and a heat-shock inducible Flippase (FLP) was Sjöström, P. J. and Van Meyel, D. J. (2014). Neuronal morphometry directly from Nat. Methods introduced either in a wild-type control (w1118)orindYPEL3T1-8 mutants bitmap images. 11, 982-984. doi:10.1038/nmeth.3125 ppk-GAL4 Gohl, D. M., Silies, M. A., Gao, X. J., Bhalerao, S., Luongo, F. J., Lin, C.-C., along with . The 3-day-old larvae grown in grape-agar plate were Potter, C. J. and Clandinin, T. R. (2011). A versatile in vivo system for directed heat shocked for 15 min in a 37°C water bath and allowed one more day dissection of gene expression patterns. Nat. Methods 8, 231-237. doi:10.1038/ of growth at 25°C before being dissected and processed for nmeth.1561 Disease Models & Mechanisms

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Gou, B., Liu, Y., Guntur, A. R., Stern, U. and Yang, C.-H. (2014). Mutlu, S., Esposti, F., Portugues, R., Borghuis, B. G., Khakh, B. S., Shigetomi, Mechanosensitive neurons on the internal reproductive tract contribute to egg- E., Filosa, A., Wardill, T. J., Takagi, R., Tian, L. et al. (2012). Optimization of a laying- induced acetic acid attraction in Drosophila. Cell Rep. 9, 522-530. doi:10. GCaMP calcium indicator for neural activity imaging. J. Neurosci. 32, 1016/j.celrep.2014.09.033 13819-13840. doi:10.1523/JNEUROSCI.2601-12.2012 Grueber, W. B., Ye, B., Yang, C.-H., Younger, S., Borden, K., Jan, L. Y. and Jan, Nern, A., Pfeiffer, B. D., Svoboda, K. and Rubin, G. M. (2011). Multiple new site- Y.-N. (2007). Projections of Drosophila multidendritic neurons in the central specific recombinases for use in manipulating animal . Proc. Natl. Acad. nervous system: links with peripheral dendrite morphology. Development 134, Sci. USA 108, 14198-14203. doi:10.1073/pnas.1111704108 55-64. doi:10.1242/dev.02666 Lin, C.-C. and Potter, C. J. (2016). Editing transgenic DNA components by Han, J. H., Shin, J. H., Lee, Y. H. and Kim, K. S. (2018). Distinct roles of the YPEL inducible gene replacement in Drosophila melanogaster. Genetics 203, gene family in development and pathogenicity in the ascomycete fungus 1613-1628. doi:10.1534/genetics.116.191783 Magnaporthe oryzae. Sci. Rep. 8, 1-15. doi:10.1038/s41598-018-32633-6 Ohyama, T., Schneider-Mizell, C. M., Fetter, R. D., Aleman, J. V., Franconville, Hattori, Y., Sugimura, K. and Uemura, T. (2007). Selective expression of Knot/ R., Rivera-Alba, M., Mensh, B. D., Branson, K. M., Simpson, J. H., Truman, Collier, a transcriptional regulator of the EBF/Olf-1 family, endows the Drosophila J. W. et al. (2015). A multilevel multimodal circuit enhances action selection in Nature sensory system with neuronal class-specific elaborated dendritic patterns. Genes Drosophila. 520, 633-639. doi:10.1038/nature14297 Cells 12, 1011-1022. doi:10.1111/j.1365-2443.2007.01107.x Pfeiffer, B. D., Ngo, T. T. B., Hibbard, K. L., Murphy, C., Jenett, A., Truman, J. W. Honjo, K., Hwang, R. Y. and Tracey, W. D. (2012). Optogenetic manipulation of and Rubin, G. M. (2010). Refinement of tools for targeted gene expression in Genetics neural circuits and behavior in Drosophila larvae. Nat. Protoc. 7, 1470-1478. Drosophila. 186, 735-755. doi:10.1534/genetics.110.119917 doi:10.1038/nprot.2012.079 Port, F., Chen, H.-M., Lee, T. and Bullock, S. L. (2014). Optimized CRISPR/Cas Proc. Hosono, K., Sasaki, T., Minoshima, S. and Shimizu, N. (2004). Identification and tools for efficient germline and somatic engineering in Drosophila. Natl. Acad. Sci. USA characterization of a novel gene family YPEL in a wide spectrum of eukaryotic 111, E2967-E2976. doi:10.1073/pnas.1405500111 species. Gene 340, 31-43. doi:10.1016/j.gene.2004.06.014 Shearin, H. K., Dvarishkis, A. R., Kozeluh, C. D. and Stowers, R. S. (2013). PLoS ONE Hu, Y., Flockhart, I., Vinayagam, A., Bergwitz, C., Berger, B., Perrimon, N. and Expansion of the gateway MultiSite recombination cloning toolkit. 8, Mohr, S. E. (2011). An integrative approach to ortholog prediction for disease- e77724. doi:10.1371/journal.pone.0077724 Sholl, D. A. (1953). Dendritic organization in the neurons of the visual and motor focused and other functional studies. BMC Bioinformatics. 12, 357. doi:10.1186/ cortices of the cat. J. Anat. 87, 387-406. doi:10.1038/171387a0 1471-2105-12-357 Soldano, A., Alpizar, Y. A., Boonen, B., Franco, L., López-Requena, A., Liu, G., Hwang, R. Y., Zhong, L., Xu, Y., Johnson, T., Zhang, F., Deisseroth, K. and Mora, N., Yaksi, E., Voets, T., Vennekens, R., (2016). Gustatory-mediated Tracey,W.D.(2007). Nociceptive neurons protect Drosophila larvae from avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila. parasitoid wasps. Curr. Biol. 17, 2105-2116. doi:10.1016/j.cub.2007.11.029 Elife 5, e13133. doi:10.7554/eLife.13133 Jenett, A., Rubin, G. M., Ngo, T. T. B., Shepherd, D., Murphy, C., Dionne, H., Stewart, B. A., Atwood, H. L., Renger, J. J., Wang, J. and Wu, C.-F. (1994). Pfeiffer, B. D., Cavallaro, A., Hall, D., Jeter, J. et al. (2012). A GAL4-driver line Improved stability of Drosophila larval neuromuscular preparations in resource for Drosophila neurobiology. Cell Rep. 2, 991-1001. doi:10.1016/j. haemolymph-like physiological solutions. J. Comp. Physiol. A 175, 179-191. celrep.2012.09.011 doi:10.1007/BF00215114 Jinushi-Nakao, S., Kinameri, E., Liu, A. W., Arvind, R., Moore, A. W. and Subramanian, L., Medina-Pritchard, B., Barton, R., Spiller, F., Kulasegaran- Amikura, R. (2007). Knot/Collier and cut control different aspects of dendrite Shylini, R., Radaviciute, G., Allshire, R. C. and Jeyaprakash, A. A. (2016). Neuron cytoskeleton and synergize to define final arbor shape. 56, 963-978. Centromere localization and function of Mis18 requires Yippee-like domain- doi:10.1016/j.neuron.2007.10.031 mediated oligomerization. EMBO Rep. 17, 496-507. doi:10.15252/embr. Kaneko, T., Macara, A. M., Li, R., Hu, Y., Iwasaki, K., Dunnings, Z., Firestone, E., 201541520 Horvatic, S., Guntur, A., Shafer, O. T. et al. (2017). Serotonergic modulation Wong, A. M., Wang, J. W. and Axel, R. (2002). Spatial representation of the enables pathway-specific plasticity in a developing sensory circuit in Drosophila. glomerular map in the Drosophila protocerebrum. Cell 109, 229-241. doi:10.1016/ Neuron 95, 623-638. doi:10.1016/j.neuron.2017.06.034 S0092-8674(02)00707-9 Kelley, K. D., Miller, K. R., Todd, A., Kelley, A. R., Tuttle, R. and Berberich, S. J. Xiang, Y., Yuan, Q., Vogt, N., Looger, L. L., Jan, L. Y. and Jan, Y. N. (2010). Light- Cancer (2010). YPEL3, a p53-regulated gene that induces cellular . avoidance-mediating photoreceptors tile the Drosophila larval body wall. Nature Res. 70, 3566-3575. doi:10.1158/0008-5472.CAN-09-3219 468, 921-926. doi:10.1038/nature09576 Kim, J. H., Wang, X., Coolon, R. and Ye, B. (2013). Dscam expression levels Yang, L., Li, R., Kaneko, T., Takle, K., Morikawa, R. K., Essex, L., Wang, X., determine presynaptic arbor sizes in drosophila sensory neurons. Neuron 78, Zhou, J., Emoto, K., Xiang, Y. et al. (2014). Trim9 regulates activity-dependent 827-838. doi:10.1016/j.neuron.2013.05.020 fine-scale topography in drosophila. Curr. Biol. 24, 1024-1030. doi:10.1016/j.cub. Longair, M. H., Baker, D. A. and Armstrong, J. D. (2011). Simple neurite tracer: 2014.03.041 open source software for reconstruction, visualization and analysis of neuronal Zhang, J., Wen, X., Ren, X.-Y., Li, Y.-Q., Tang, X.-R., Wang, Y.-Q., He, Q.-M., processes. Bioinformatics 27, 2453-2454. doi:10.1093/bioinformatics/btr390 Yang, X.-J., Sun, Y., Liu, N. et al. (2016). YPEL3 suppresses epithelial- Macpherson, L. J., Zaharieva, E. E., Kearney, P. J., Alpert, M. H., Lin, T.-Y., mesenchymal transition and metastasis of nasopharyngeal carcinoma cells Turan, Z., Lee, C.-H. and Gallio, M. (2015). Dynamic labelling of neural through the Wnt/β-catenin signaling pathway. J. Exp. Clin. Cancer Res. 35, 1-10. connections in multiple colours by trans-synaptic fluorescence complementation. doi:10.1186/s13046-015-0276-9 Nat. Commun. 6, 10024. doi:10.1038/ncomms10024 Zhong, L., Bellemer, A., Yan, H., Honjo, K., Robertson, J., Hwang, R. Y., Pitt, Mohammad, F., Stewart, J. C., Ott, S., Chlebikova, K., Chua, J. Y., Koh, T.-W., G. S. and Tracey, W. D. (2012). Thermosensory and nonthermosensory isoforms Ho, J. and Claridge-Chang, A. (2017). Optogenetic inhibition of behavior with of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP anion channelrhodopsins. Nat. Methods 14, 271-274. doi:10.1038/nmeth.4148 channel. Cell Rep. 1, 43-55. doi:10.1016/j.celrep.2011.11.002 Disease Models & Mechanisms

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