RESEARCH ARTICLE

Frameshift mutations of YPEL3 alter the sensory circuit functionin DrosophilaJung Hwan Kim1,*, Monika Singh1, Geng Pan2, Adrian Lopez1, Nicholas Zito1, Benjamin Bosse1 andBing Ye2,*

ABSTRACTA frameshift mutation inYippee-like (YPEL) 3was recently found froma rare human disorder with peripheral neurological conditionsincluding hypotonia and areflexia. The YPEL gene family is highlyconserved from yeast to human, but its members’ functions are poorlydefined. Moreover, the pathogenicity of the human YPEL3 variant iscompletely unknown. We generated a Drosophila model of humanYPEL3 variant and a genetic null allele of Drosophila homolog ofYPEL3 (referred to as dYPEL3). Gene-trap analysis suggests thatdYPEL3 is predominantly expressed in subsets of neurons, includinglarval nociceptors. Analysis of chemical nociception induced by allyl-isothiocyanate (AITC), a natural chemical stimulant, revealedreduced nociceptive responses in both dYPEL3 frameshift and nullmutants. Subsequent circuit analysis showed reduced activation ofsecond-order neurons (SONs) in the pathway without affectingnociceptor activation upon AITC treatment. Although the grossaxonal and dendritic development of nociceptors was unaffected,the synaptic contact between nociceptors and SONs was decreasedby the dYPEL3mutations. Furthermore, expressing dYPEL3 in larvalnociceptors rescued the behavioral deficit in dYPEL3 frameshiftmutants, suggesting a presynaptic origin of the deficit. Together,these findings suggest that the frameshift mutation results in YPEL3loss of function and may cause neurological conditions by weakeningsynaptic connections through presynaptic mechanisms.

KEY WORDS: YPEL3, Pathogenicity, Rare mutation, Synapticconnection

INTRODUCTIONYPEL3 belongs to the Yippee-like gene family, which is composedof a number of genes in eukaryotic species ranging from yeast tohuman (Hosono et al., 2004). Only a handful of studies have hintedat the biological roles of YPEL3. YPEL3 was initially identified as asmall unstable apoptotic protein because of its low protein stabilityand the ability to induce apoptosis when overexpressed in a myeloidcell line (Baker, 2003). Subsequent studies implicated YPEL3 as atumor suppressor. YPEL3 expression correlates with p53 activity(Kelley et al., 2010). Overexpression and knockdown analyses

suggest that YPEL3 suppresses the epithelial-to-mesenchymaltransition in cancer cell lines by increasing GSK3β expression(Zhang et al., 2016). Other studies have shown the role of YPELgenes in development. The loss-of-function mutations of YPELorthologs in ascomycete fungus altered fungal conidiation andappressoria development (Han et al., 2018). In zebrafish, amorpholino-mediated targeting of ypel3 altered brain structures(Blaker-Lee et al., 2012).

Recently, a mutation in human YPEL3was found in a patient witha rare disorder that manifests a number of neurological symptoms[the National Institutes of Health (NIH)-Undiagnosed DiseasesProgram]. The mutation was caused by duplication of a nucleotidein a coding exon of YPEL3, resulting in a frameshift andconsequently a premature stop codon. The clinical observationshowed that the patient had normal cognition but manifestedperipheral symptoms, including areflexia and hypotonia. However,whether the identified YPEL3mutation is pathogenic in the nervoussystem is unknown. Moreover, little is known about the functions ofYPEL3 in the nervous system.

In the present study, we generated a Drosophila model of thehuman condition by creating the disease-relevant YPEL3 variantusing CRISPR/Cas9-mediated in-del mutations. Our gene-trapanalysis suggests that subsets of neurons, including nociceptors,express theDrosophila homolog of YPEL3 (referred to as dYPEL3).Subsequent analysis revealed reduced nociceptive behavior indYPEL3 mutants. Consistently, we found that dYPEL3 mutationsimpaired the activation of second-order neurons (SONs) in thenociceptive pathway and reduced the synaptic contact betweennociceptors and these SONs. We further demonstrate that thebehavioral, circuit and cellular phenotypes in the dYPEL3frameshift mutants are recapitulated in a genetically null allele ofdYPEL3, and that expressing wild-type dYPEL3 in nociceptorsrescues the altered nociceptive behavior in the frameshift mutants.These findings suggest that the identified human YPEL3mutation ispathogenic and affects neuronal synapses through a loss-of-functionmechanism.

RESULTSGeneration of a disease-relevant variant of YPEL3 inDrosophilaAlthough the discovery of a YPEL3 variant in a patient underscoresthe importance of YPEL3 in human health, whether this variantcauses any deficits in the nervous system is unknown. There are fiveYPEL genes in human: YPEL1-YPEL5. YPEL1, YPEL2, YPEL3and YPEL4 are highly homologous to each other (up to 96%identity at amino acid sequences), whereas YPEL5 has only ∼40%homology to the other members (Hosono et al., 2004). We foundtwo YPEL homologs inDrosophila, Yippee andCG15309, using anortholog search (Hu et al., 2011). The predicted amino acidsequences of CG15309 showed 88% similarity (81% identity) to

Handling Editor: Steven J. ClapcoteReceived 12 September 2019; Accepted 31 March 2020

1Department of Biology, University of Nevada, Reno, Reno, NV 89557, USA. 2LifeSciences Institute and Department of Cell and Developmental Biology, University ofMichigan, Ann Arbor, MI 48109, USA.

*Authors for correspondence ( jungkim@unr.edu; bingye@umich.edu)

J.H.K., 0000-0001-8548-4435; B.Y., 0000-0002-8828-4065

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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human YPEL3 (Fig. 1A), while that of Yippee showed 65%similarity (53% identity) (data not shown). Yippee appears to be anortholog of YPEL5 because it is more closely related to YPEL5 thanYPEL3, with 87% similarity and 73% identity to YPEL5 (data notshown). Therefore, we named CG15309 as dYPEL3.The variant identified in the human patient introduces an extra

nucleotide in the middle of the coding exon, which produces aframeshift and consequently results in the incorporation of the 37ectopic amino acids followed by a premature stop codon (Fig. 1B).To generate a Drosophila model of the human variant, we tookadvantage of the CRISPR/Cas9 technology to induce in-delmutations (Port et al., 2014). The entire coding sequence ofdYPEL3 is in a single exon. We designed a guide RNA that targetsthe middle of the coding exon (Fig. 1C, top) and successfullyisolated two dYPEL3 frameshift mutants named dYPEL3T1-6 anddYPEL3T1-8 (Fig. 1C, middle). dYPEL3T1-6 has a two-nucleotidedeletion at 121 nucleotides downstream of a start codon, whichgenerated a premature stop codon at 153 nucleotides downstream ofa start codon, while dYPEL3T1-8 carries a four-nucleotide deletion at118 nucleotides downstream of a start codon, and generated apremature stop codon at 145 nucleotides downstream of a startcodon. Similar to the human variant, the mutations introducedadditional amino acids followed by a premature stop codon (Fig. 1C,middle). The ectopic amino acids in dYPEL3T1-6 closely resemblethose of the human variant (Fig. 1C, bottom).

dYPEL3 is expressed in subsets of neuronsWe did not find any gross developmental defects in dYPEL3T1-6 ordYPEL3T1-8 flies. Homozygotes were viable and fertile, and showednormal growth under standard culture condition (data not shown).This raises the possibility that dYPEL3 is expressed in a subset ofcells in the body. Our efforts of generating antibodies againstdYPEL3 failed in two independent trials, precluding the use ofimmunostaining for identifying the cell types that express dYPEL3.We thus took advantage of a GAL4 enhancer-trap line, CG15309-GAL4 (dYPEL3-GAL4) (Gohl et al., 2011), to study the expressionpattern of dYPEL3 in flies. This line contains a GAL4 insertion inthe first intron of dYPEL3, which places the GAL4 under the controlof the endogenous dYPEL3 promoter and enhancers (Fig. 2A, top).We expressed a membrane GFP reporter (mouse CD8::GFP ormCD8::GFP) to visualize dYPEL3 expression pattern inDrosophilalarvae. A small number of cells in the larval central nervous system(CNS), including the ventral nerve cord and brain, were labeled bymCD8::GFP (Fig. 2A, bottom). These cells extended fine processesthat cover most of the neuropil area in the larval CNS, suggestingthat they are neurons. To identify the cell types that expressdYPEL3, dYPEL3-GAL4>mCD8::GFP samples were co-immunostained with the neuron marker anti-Elav and the glialmarker anti-Repo (Fig. 2B). Approximately 85% of cells that werelabeled with dYPEL3-GAL4 were positive for Elav, but none werepositive for Repo (Fig. 2C). This result suggests that dYPEL3 ispredominantly expressed in neurons, but not in glia. Interestingly,dYPEL3-GAL4 also labeled a subset of sensory neurons, includingthe class IV dendritic arborization (da) neurons (nociceptors), classIII da neurons and chordotonal neurons (both mechanosensors), butnot the class I da neurons (proprioceptors) (Fig. 2Bii,iii; Fig. S1).dYPEL3 was not expressed in muscles or epidermal cells (Fig. S2).

The disease-relevant mutations of dYPEL3 reducenociceptive behavioral responsesThe human patient shows symptoms mainly in the peripheralnervous system (PNS), including areflexia and hypotonia (the

NIH-Undiagnosed Diseases Program). We thus focused ouranalysis on dYPEL3-positive neurons in the PNS (Fig. 2Bii,iii).The enhancer-trap analysis suggests that nociceptors expressdYPEL3. This finding was confirmed by co-immunostaining withthe nociceptor marker anti-Knot antibody (Hattori et al., 2007;Jinushi-Nakao et al., 2007) (Fig. 3A). The nociceptors detectvarious stimuli, including noxious heat, touch and chemicals, andactivate the nociceptive pathway that leads to the nocifensive rollingbehavior (Hwang et al., 2007). Allyl-isothiocyanate (AITC), anatural chemical stimulant, has been proposed to cause larvalnociceptive behavior through nociceptors (Kaneko et al., 2017;Xiang et al., 2010; Zhong et al., 2012). Since AITC also elicitsnociceptive behavior in adult flies through gustatory sensoryneurons (Soldano et al., 2016), we determined whether AITC-induced nocifensive rolling required the larval nociceptors.Optogenetic inhibition of larval nociceptors with Guillardia thetaanion channel rhodopsin-1 (GtACR1) (Mohammad et al., 2017)dramatically reduced AITC-induced rolling (Fig. S3),demonstrating that the AITC-induced rolling depends on thenociceptors on the larval body wall.

We first determined whether the functions of nociceptors werealtered by the dYPEL3mutations. We applied AITC to thewild-typecontrol, dYPEL3T1-6 and dYPEL3T1-8 and found a significantreduction in nociceptive rolling behavior in the dYPEL3 mutants(48% and 40% reduction, respectively, Fig. 3B). The extent ofdecrease in nociceptive rolling was not different between the twomutant alleles of dYPEL3, which are almost identical except for thesequences in the ectopic stretch of amino acids (Fig. 1C). Thissuggests that the truncation of dYPEL3, but not the presence of theectopic amino acid sequences, is responsible for the observedphenotype. dYPEL3T1-8 represents a simpler version since it onlyhas incorporation of a few ectopic amino acids (Fig. 1C). Therefore,we focused our analysis on dYPEL3T1-8 for further analysis.

How does dYPEL3mutation affect the sensory function? We firstlooked into whether the dYPEL3 mutation affects the developmentof nociceptors. We expressed mCD8::GFP specifically innociceptors in wild-type and dYPEL3T1-8 larvae using thenociceptor-specific driver ppk-GAL4 (Grueber et al., 2007). Thedendritic arborization was assessed using Sholl analysis (Sholl,1953) and by measuring total dendritic length. dYPEL3T1-8

mutations did not alter the dendritic development (Fig. 4A,B).Next, we tested whether the presynaptic terminals of nociceptors aredefective in dYPEL3 mutants. To this end, a flip-out mosaicexperiment was performed to label single nociceptive presynapticarbors (Yang et al., 2014). The total length of the presynaptic arborof each nociceptor was indistinguishable between wild type anddYPEL3T1-8 (Fig. 4B), suggesting that dYPEL3T1-8 does not affectthe development of presynaptic arbors.

The disease-relevant mutations of dYPEL3 reduce thesynaptic transmission from nociceptors to theirpostsynaptic neuronsNext, we assessed the synaptic transmission from nociceptors totheir postsynaptic neuron Basin-4, a key SON in the nociceptivepathway (Ohyama et al., 2015). The activation of Basin-4 elicitsnociceptive behavior even in the absence of nociceptor activation,while silencing these neurons suppresses nociceptive behavior(Ohyama et al., 2015). The genetically encoded calcium indicatorGCaMP6f was selectively expressed in Basin-4 for recordingintracellular calcium, a proxy of neuronal activity (Chen et al., 2013)(Fig. 5A). Larvae were dissected in insect saline as a filletpreparation with intact PNS and CNS (Kaneko et al., 2017) and

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treated with AITC to stimulate the nociceptors. AITC elicited robustGCaMP signals that persisted over several minutes in bothnociceptors and Basin-4 neurons (Fig. 5A,B). We found that theGCaMP signals in Basin-4 neurons were significantly decreased in

dYPEL3T1-8 mutants, compared to wild-type control (Fig. 5A, 43%decrease). By contrast, GCaMP measurement in nociceptor axonterminals showed that dYPEL3T1-8 did not change AITC-inducedactivation of nociceptors (Fig. 5B).

Fig. 1. The generation of aDrosophilamodel of YPEL3 frameshift mutation. (A)CG15309 is theDrosophila homolog of human YPEL3. Sequence alignmentbetween human YPEL3 (YPEL3) and Drosophila CG15309. Shaded in pink are the identical amino acid sequences. (B) Duplication of a cytosine nucleotide inYPEL3 gene from a patient (top). A predicted molecular lesion in human YPEL3 (bottom) introduces an ectopic amino acid sequence (shaded in green). Thepreserved region is shaded in pink. (C) CRISPR-Cas9mediated in-del mutation inCG15309/dYPEL3. A guide RNA is designed targeting themiddle of the codingexon (top). The isolated dYPEL3 in-del mutants (middle). Sequence alignment between wild type (wt), dYPEL3T1-6 and dYPEL3T1-8. The introduced ectopicamino acid sequences following a premature stop codon are shaded in green. The sequence alignment of the introduced ectopic amino acid sequences from thehuman YPEL3 frameshift mutants and dYPEL3T1-6 (bottom). The identical amino acid sequences are shaded in pink. ORF, open reading frame.

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The disease-relevant mutations of dYPEL3 reduce thesynaptic contact between nociceptors and theirpostsynaptic neuronsHow do the dYPEL3 mutations reduce the nociceptor-to-Basin-4synaptic transmission? To address this, we employed a synaptic-contact-specific GFP reconstitution across synaptic partners (GRASP)technique, termed syb-GRASP (Macpherson et al., 2015), to assess

the synaptic contact between the presynaptic terminals of nociceptorsand the dendrites of Basin-4 neurons. The GRASP technique utilizestwo separate fragments of GFP molecule – split-GFP1-10 (spGFP1-10) and split-GFP11 (spGFP11), which can be detected by a specificanti-GFP antibody only when the two fragments are in closeproximity to reconstitute a complete GFP. In syb-GRASP, spGFP1-10 is fused to the synaptic vesicle protein Synaptobrevin andexpressed in the presynaptic neurons, whereas spGFP11 is fused to ageneral membrane tag and expressed in postsynaptic neurons. Twoindependent binary gene expression systems,GAL4-UAS and LexA-LexAop, were used to drive the expression of spGFP1-10 andspGFP11 in different cell types (del Valle Rodríguez et al., 2012).Synaptic vesicle exocytosis from presynaptic terminals exposesspGFP1-10 onto the presynaptic cleft, where it reconstitutes thefunctional GFPmolecule by associating with postsynaptic spGFP11molecules. This technique has been used widely to visualizesynaptic contact between two identified neuron types.

The spGFP1-10 and spGFP11 were specifically expressed innociceptors and Basin-4 neurons, respectively (Fig. 6A, left). Theresulting GRASP signal was measured in each segmental neuropil,and normalized by the spGFP1-10 intensity in wild type and indYPEL3T1-8 (Fig. 6A, right). We detected a mild, but significant,decrease (23%) in the GRASP signals in dYPEL3T1-8, compared tothose in wild-type control (Fig. 6B). This suggests that the synapticcontact between nociceptors and its synaptic target Basin-4 iscompromised in dYPEL3T1-8.

Fig. 2. dYPEL3 is a neuronal gene. (A) The expression pattern of dYPEL3 inthe CNS. The InSITE gene trap line for dYPEL3 was used (CG15309-GAL4/dYPEL3-GAL4). GAL4 transcription factor is inserted in the first intron. Theintroduction of UAS-mCD8::GFP demonstrates the endogenous expressionpattern of dYPEL3. Note that the CG15309-GAL-positive cells elaborate fineprocesses throughout the CNS. Scale bar: 50 µm. (B) mCD8::GFP (magenta)was expressed under dYPEL3-GAL4 following immunostaining with anti-Elav(neuronal, green) and anti-Repo (glial, blue) antibodies. (i) The CNS. Cellbodies in 1 and 2 are shown magnified on the top right. (ii,iii) Chordotonalneurons (ii, arrows) and a class III da neuron (iii) in the PNS. iii also shows aclass IV da neuron (nociceptor) that is positive for dYPEL3 (arrow). Scale bar:10 µm. (C) Quantitation of the Elav-positive and Repo-positive cells that arelabeled with CG15309-GAL4. The majority of dYPEL3-postive cells were Elavpositive, but none were positive for Repo.

Fig. 3. dYPEL3 frameshift mutations reduce nociceptive behavior.(A) Nociceptive/class IV da neurons are positive for dYPEL3. A nuclearGFP (GFP-nls, green) was expressed under dYPEL3-GAL4 followingimmunostaining with anti-Knot antibody (blue). Anti-HRP antibody was used tolabel all PNS neurons (magenta). Scale bar: 10 μm. (B) The AITC-inducednociceptive behavior was measured in a wild-type control (wt) and dYPEL3frameshift mutants (dYPEL3T1-8 and dYPEL3T1-6). The number of larvae thatexhibited complete rolling behavior was scored and expressed as apercentage (n=252 for each genotype). The Chi-squared test was performedbetween the groups. NS, non-significant; ****P<0.0001.

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The disease-relevant frameshift mutant of dYPEL3 is aloss-of-function alleleThe mutations in the patient and in our Drosophilamodel introducepremature stop codons, which may induce the nonsense-mediateddecay (Chang et al., 2007), resulting in YPEL3 loss of function.However, the frameshift mutation in YPEL3 may escape fromnonsense-mediated decay because the premature stop codons are inthe last coding exons (both in human and Drosophila), which maylead to the production of a truncated version of YPEL3 proteins. Todiscern these possibilities, we generated a genetically null allele ofdYPEL3 (dYPEL3KO) by removing the entire dYPEL3 coding regionusing CRISPR/Cas9 (Fig. 7A). We found that dYPEL3KO larvaerecapitulated all the deficits found in dYPEL3 frameshift mutants tothe similar extent. These include AITC-induced rolling behavior(40% decrease), AITC-induced Basin-4 activation (47% decrease),and synaptic contact between nociceptors and Basin-4 (24%decrease) (Fig. 7B-D). These results strongly suggest that thedisease-relevant mutation of dYPEL3, dYPEL3T1-8, is a loss-of-function allele.If dYPEL3 frameshift mutation is loss of function, the defects in

these mutants may be rescued by the expression of wild-typedYPEL3. The nociceptors, but not Basin-4 neurons, express dYPEL3(Fig. 3A; Fig. S4). Thus, we expressed dYPEL3 specifically innociceptors using ppk-GAL4 (Grueber et al., 2007) in wild-type anddYPEL3T1-8 larvae and tested AITC-induced nociceptive behavior.We found that expressing dYPEL3 in dYPEL3T1-8 completely

rescued defective rolling behavior induced by AITC (Fig. 8),whereas expressing dYPEL3 in wild type had no effect.

Taken together, these results strongly support a model that theframeshift in human YPEL3 causes YPEL3 loss of function, and thatYPEL3 acts in presynaptic neurons to positively regulate synapticcontact.

DISCUSSIONThe biological functions of the YPEL gene family, includingYPEL3, are poorly understood. Moreover, whether the identifiedYPEL3 frameshift mutation is pathogenic is unknown. Drosophilaprovides a powerful tool for analyzing disease-relevant human genemutations (Bellen et al., 2019). In this study, we report aDrosophilamodel of human YPEL3 mutation and demonstrate that the disease-relevant YPEL3 frameshift mutations are pathogenic in the nervoussystem.

The YPEL gene family is highly conserved across eukaryotesranging from yeast to human. Likewise, our homology analysisindicated a strikingly high homology in gene sequences betweenhuman and Drosophila YPEL3 (80% identity, Fig. 1B).Interestingly, it appears that the sequence homology extends evento the nucleotide level since the analogous frameshift mutation gaverise to the generation of similar amino acid sequences in the ectopicsequences in dYPEL3T1-6 (Fig. 1C). Given such high sequencehomology, we envision that the functions of human YPEL3 andDrosophila YPEL3 are also conserved. The YPEL family can be

Fig. 4. The development of nociceptors isnot altered by dYPEL3 frameshiftmutations. (A) mCD8::GFP was specificallyexpressed in nociceptors using ppk-GAL4 inwild-type control (wt) and dYPEL3 frameshiftmutants (dYPEL3T1-8). Total length ofdendrites was measured (n=6 for eachgenotype). Unpaired Student’s t-test withWelch’s correction was performed. Scalebar: 50 µm. (B) Sholl analysis wasperformed with 20-µm radius increment fromthe dendritic tracing. Number of totalcrossings within 300 µm from the cell centerwas measured. Two-way ANOVA for Shollanalysis and unpaired Student’s t-test withWelch’s correction for total dendriticcrossings were performed. (C) The axonterminals of single nociceptors from wildtype and dYPEL3T1-8 mutants werevisualized using the flip-out technique. Thetotal length of axon terminals was measured(n=12 for wt, n=14 for dYPEL3T1-8). Scalebar: 10 µm. Unpaired Student’s t-test withWelch’s correction was performed. Data arepresented as mean±s.e.m. All statisticalanalysis was two-tailed. NS, non-significant.

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subdivided into two categories. Human YPEL1, YPEL2, YPEL3and YPEL4 belong to one with high homology with each other,while YPLE5 constitutes a distinct family (Hosono et al., 2004). InDrosophila, there is only a single homolog of human YPEL1-YELP4, CG15309 (Fig. 1B). Because the tissue expression patternsof YPEL genes are complex in human and mice (Hosono et al.,2004), the single YPEL gene makes Drosophila advantageous as amodel for studying YPEL3-induced pathogenesis.

In human and mice, YPEL3 is ubiquitously expressed, as basedon results from RT-PCR experiments (Hosono et al., 2004).Northern blot analysis of murine tissues shows relative enrichmentof YPEL3 in brain and liver tissue (Baker, 2003). Our results basedon a gene-trap Drosophila line indicates that dYPEL3 is expressedin subsets of neurons, but not in glia (Fig. 2B,C). The human patientexhibited multiple neurological symptoms in the PNS, but hadnormal cognition (the NIH-Undiagnosed Diseases Program).Interestingly, dYPEL3-GAL4 was selectively expressed innociceptors and mechanosensors in the PNS (Fig. 3A; Fig. S2).Furthermore, YPEL3 frameshift mutations reduced nociceptivebehavior (Fig. 3B). These results suggest that at least some of theneurological symptoms in the human patient originate from neuronsthat express YPEL3.

How does the YPEL3 frameshift mutation cause sensory deficits?The gross neuronal development of nociceptors was not altered bythe dYPEL3 mutations (Fig. 4). Calcium-imaging experimentsshowed that activation of nociceptors by AITC was not altered in

Fig. 5. dYPEL3 frameshift mutations reduce the synaptic transmissionfrom nociceptors to Basin-4 neurons. (A) Basin-4 activation upon AITCtreatment was reduced by dYPEL3T1-8. GCaMP6f was expressed in Basin-4neurons. Nociceptors were activated with 10 mMAITC (top left). Ca2+ increasein Basin-4 was measured by GCaMP fluorescence and the tracing over time isshown (n=40 for wt, n=47 for dYPEL3T1-8) (top right). The cumulative GCaMPactivation from single Basin-4 neurons was measured and presented as mean±s.e.m. (bottom left), as well as in a violin plot to show distribution (bottom right).Mann–Whitney test. (B) Nociceptor activation was not altered by dYPEL3mutations. GCaMP6f was expressed in nociceptors using ppk-GAL4.Nociceptorswere activatedwith 10 mMAITC (top left). Ca2+ increase in the axonterminals of nociceptors wasmeasured byGCaMP fluorescence and the tracingover time is shown (n=13 for each genotype) (top right). The cumulative GCaMPactivation from the nociceptor axon terminals was measured and presented asmean±s.e.m. (bottom left), as well as in a violin plot to show distribution (bottomright). Mann–Whitney test. All statistical analysis was two-tailed. NS, non-significant; ****P<0.0001.

Fig. 6. dYPEL3 frameshift mutations reduce the synaptic contact betweennociceptors and Basin-4 neurons. (A) The syb-GRASP technique was usedto report the synaptic contact between nociceptors and Basin-4. The spGFP1-10 (red cylinders) and spGFP11 (blue sectors) were expressed in nociceptorsand Basin-4, respectively (left). The resulting GRASP signal was visualized byanti-GRASP antibody (green), and the spGFP1-10 that is expressed innociceptor axon terminals was used as a normalization control (magenta)(right). Scale bar: 10 μm. (B) The GRASP intensity from each neuropil wasnormalized 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.

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dYPEL3T1-8 mutants (Fig. 5B). Rather, dYPEL3T1-8 reduced Basin-4 responses to nociceptor stimulation (Fig. 5A). This suggests thatthe neurotransmission from nociceptors to their postsynapticneurons is reduced by the dYPEL3 frameshift mutation. Thisconclusion is corroborated by the finding that the syb-GRASPsignal between nociceptors and Basin-4 was reduced (Fig. 6). Sincethe syb-GRASP technique requires synaptic release (Macphersonet al., 2015), it is possible that the decrease in syb-GRASP signalsreflects reduced activity or synaptic release in nociceptors in themutants. Alternatively, the reduction in syb-GRASP signals mightbe due to a reduced number of synapses in the mutants. Additionaltechniques are needed to discern these possibilities. Nevertheless,the results from calcium imaging and syb-GRASP experimentsconsistently show reduced synaptic transmission from nociceptorsto the SON Basin-4. Because Basin-4 activation is central to

nociceptive behavior (Ohyama et al., 2015), the reduced synaptictransmission from nociceptors to Basin-4 is likely responsible forthe reduction in nociceptive behavior in dYPEL3 mutants. It isintriguing that the human patient has peripheral symptoms ofhypotonia and areflexia; both may arise from reduced synaptictransmission.

We observed that AITC-elicited GCaMP signals persisted overa fewminutes in both nociceptors and Basin-4 neurons (Figs 5A,Band 7C). Since AITC does not induce continuous larva rollingover such a long period, this implies the presence of an acuteadaptation to AITC stimulation in the nociceptive circuit. It ispossible that the ex vivo GCaMP measurement does not fullyrecapitulate neural activity in vivo. Nevertheless, our resultsindicate that the dYPEL3 mutations significantly reduce calciumincrease in Basin-4 neurons.

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-basedgenotyping. Note that dYPEL3KO showed the PCR amplifications specific for DsRed-cassette, but not the ones from wild type. (B) dYPEL3 knockoutreduces 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 inBasin-4 neurons. Nociceptors were activated with 10 mM AITC. Ca2+ increase in Basin-4 was measured by GCaMP fluorescence and the GCaMP traceover 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 asmean±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 signalsbetween nociceptors and Basin-4. Synaptobrevin-spGFP1-10 and spGFP11 were expressed in nociceptors and Basin-4, respectively. The resulting GRASPsignal was visualized by anti-GFP antibody that only recognizes the reconstituted GFP (anti-GRASP) (green), and the spGFP1-10 expressed in nociceptor axonterminals was used as a normalization control (magenta) (left). Scale bar: 10 μm. The GRASP intensity from each neuropil was normalized by spGFP1-10intensity 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–Whitneytest. ***P<0.001 and ****P<0.0001.

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How does the YPEL3 frameshift mutation affect YPEL3 genefunction? Our results suggest that YPEL3 frameshift mutations causeloss of function. The behavioral, circuit and synaptic phenotypeswerealmost identical between dYPEL3 frameshift (dYPEL3T1-8) andknockout (dYPEL3KO) mutants (Figs 3, 5, 6 and 7). Sincenociceptors, but not Basin-4, express dYPEL3 (Fig. 3A; Fig. S4),dYPEL3 mutations likely affect presynaptic functions. Consistently,the behavioral phenotype in dYPEL3T1-8 was completely rescued byexpressing wild-type dYPEL3 in nociceptors (Fig. 8). Takentogether, these findings suggest that YPEL3 functions inpresynaptic neurons to regulate synaptic transmission, and thatframeshift mutations in YPEL3 result in loss of YPEL3.The molecular function of YPEL3 is unclear. It contains

predicted zinc-finger motifs (Hosono et al., 2004). The zinc-finger motifs in a YPEL domain of the yeast protein Mis18 isimportant for the folding of the YPEL domain, which mediates thecentromeric localization of Mis18 (Subramanian et al., 2016). TheYPEL domain in Mis18 has ∼20% sequence similarity to YPELproteins. Since zinc-finger motifs are common in regulators of geneexpression, we suspect that the frameshift mutation of YPEL3 maychange gene expression. Indeed, overexpression of YPEL3increased the expression of GSK3β to suppress the epithelial-mesenchymal transition (Zhang et al., 2016). It is interesting to notethat GSK3β has been implicated in synaptogenesis (Cuesto et al.,2015). Thus, it will be important to determine whether YPEL3regulates the expression of genes involved in synapse formation andmaintenance and investigate how YPEL3 frameshift mutationsaffect this process.Overall, we generated a Drosophila model of the human YPEL3

frameshift mutation and found that the YPEL3 variant leads todeficits in synaptic transmission. We further demonstrate that theframeshift mutation causes loss of YPEL3 function. In addition, thisstudy establishes YPEL3 as a regulator of synaptogenesis or

maintenance. Future studies that identify the molecular mechanismsunderlying the function of YPEL3 will provide insights intotherapeutic treatments of disorders caused by YPEL3 mutations.

MATERIALS AND METHODSDrosophila melanogaster strainsDrosophila strains were kept under standard condition at 25°C in ahumidified 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 (Wonget al., 2002), hs-FLP (Nern et al., 2011) (55814), UAS-CD4-GFP (35836),UAS-GCaMP6f (Mutlu et al., 2012) (42747), LexAop-GCaMP6f (Mutluet 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 aCre-recombinase expressing fly line (1501, RRID:BDSC_1501). Thenumbers in parentheses indicate the stock numbers from the BloomingtonDrosophila Stock Center.

The generation of dYPEL3 mutantsThe CRISPR/CAS9-mediated in-del mutation was used to generate dYPEL3frameshift mutant flies. A guide RNA construct was generated in pCFD:U6:3 (Port et al., 2014) with guide RNA sequences that target the middle ofthe dYPEL3 coding exon. The standard transformation procedure wasperformed to generate a transgenic line. The transformants were crossedwith nos-Cas9 (Port et al., 2014) flies to induce in-del mutations in germcells. The resulting progeny were screened for the desired mutations by thegenomic PCR of CG15309 following the Sanger sequencing. Thegenetically null dYPEL3 allele (dYPEL3KO) was generated usingCRISPR/Cas9-mediated homology directed recombination (HDR). TheHDR donor construct was built using pBluescript as a backbone, whichincludes 3XP3:RFP (for expressing DsRed in eyes) that is flanked by loxPsequences (Lin and Potter, 2016) and two homology arms (∼700 bp and∼1 kb for right and left arms, respectively). The homology arms wereamplified from w1118 flies. Two guide RNA constructs that target near thestart and the end of dYPEL3 open reading frame (ORF) were cloned inpCFD:U6:3 (Port et al., 2014). The two guide RNA constructs and the HDRdonor construct were co-injected into w1118, nos-Cas9 fly embryos. Flieswere screened for eye expression of DsRed, and successful integration of thedonor construct was confirmed by a PCR-based genotyping. The eye-specific DsRed cassette was removed by crossing the flies carrying thedonor construct and those expressing Cre recombinase. Generation of UAS-dYPEL3 was done using pUASTattB plasmid and dYPEL3 ORF that wasamplified from w1118 genomic DNA. Standard methodology was used togenerate transformants (Bischof et al., 2007).

AITC-induced nociceptive behaviorAITC (Sigma-Aldrich) was prepared in DMSO, dissolved in water to a final25 mM concentration, and incubated on a rocker for 3 days before use. Flyembryos were grown for 5 days in a 12 h light/dark cycle at 25°C in ahumidified incubator. The third-instar larvae were moved to roomtemperature for 1 h, gently scooped out of food, washed in tap water andplaced on a grape-agar 24-well plate that had been covered with 300 µlAITC solution (25 mM). Their behavior was recorded with a digital camerafor 2 min and the number of larvae showing complete rolling behavior(minimum 360° rolling) was manually analyzed (Honjo et al., 2012). Theexperiments were paired for the wild-type control (w1118) and dYPEL3homozygous mutant larvae. Experiments were repeated three times ondifferent days with different AITC preparations. All three trials werecombined for statistical analysis.

Calcium imagingLive calcium imaging was performed using GCaMP6f (Mutlu et al., 2012).Briefly, wandering third-instar larvae – wild-type control males ordYPEL3T1-8 hemizygotes – were dissected in a modified hemolymph-like

Fig. 8. The nociceptor-specific expression of dYPEL3 in dYPEL3frameshift mutant rescues AITC-mediated larva rolling behavior.AITC-induced nociceptive behavior was measured. The nociceptor-specificexpression of dYPEL3 was achieved using ppk-GAL. The number of larvaethat exhibited complete rolling behavior was scored and expressed as apercentage (n=118 for wt, ppk>; n=115 for wt, ppk>dYPEL3; n=163 fordYPEL3T1-8, ppk>; n=103 for dYPEL3T1-8, ppk>dYPEL3). The Chi-squaredtest was performed between the groups. NS, non-significant; *P<0.05.

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3 (HL3) saline (Stewart et al., 1994) (70 mM NaCl, 5 mM KCl, 0.5 mMCaCl2, 20 mM MgCl2, 5 mM trehalose, 115 mM sucrose and 5 mMHEPES, pH 7.2). Glutamate (10 mM) was added to the HL3 solution toprevent muscle contractions and sensory feedback. The GCaMP signal wasrecorded in the entire volume of nociceptor axon terminals or Basin-4 cellbodies. Live imaging was performed with a Leica SP5 confocal system or acustom-built spinning disk microscope equipped with an extra-long-working distance 25× water objective with 2-µm step sizes. Themembrane tdTomato proteins were expressed along with GCaMP6f andused as an internal normalization control for both lateral and focus drifting.The basal GCaMP signal was recorded for a duration of 30 s to generatebaseline fluorescence (F0), and then the samples were treated with AITC(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 usingthe maximum Z-projection in ImageJ software (NIH). The region of interestwas selected in the axonal projection of nociceptors or in the cell bodies ofBasin-4. The ImageJ Time Series Analyzer plugin was used to quantify thefluorescence intensity of GCaMP6f. The cumulative GCaMPwas calculatedfrom the GCaMP tracing from AITC treatment (t=30 s) to the end ofrecording (t=180 s).

ImmunostainingImmunostaining was performed essentially as previously reported (Kimet al., 2013). The primary antibodies used were as follows: chicken anti-GFP(AB_2307313, Aves Laboratories; 1:2500), rabbit anti-RFP (600-401-379-RTU, Rockland Immunochemicals; 1:5000), rat anti-Elav (9F8A9,Developmental Studies Hybridoma Bank; 1:100) and mouse anti-Repo(8D12, Developmental Studies Hybridoma Bank; 1:5). The secondaryantibodies were from Jackson ImmunoResearch and used at 1:500 dilution:Cy2- or Cy5-conjugated goat anti-chicken, Cy2- or Cy5-conjugated goatanti-mouse, Cy5-conjugated goat anti-rabbit and Cy3-conjugated goat anti-rat. Confocal imaging was performed with a Leica SP8 confocal system or acustom-built spinning disk confocal microscope equipped with a 63× oil-immersion objective with 0.3-µm step size. The resulting 3D images wereprojected into 2D images using a maximum projection method.

In order to report the relative synaptic contact between the nociceptorsand their postsynaptic partners, syb-GRASP was performed in the malelarvae from a wild-type control (w1118), dYPEL3T1-8 or dYPEL3KO

hemizygotes. Syb::split-GFP1-10 (Macpherson et al., 2015) wasexpressed in nociceptors. CD4::split-GFP11 (Macpherson et al., 2015)was expressed in Basin-4 neurons. The polyclonal chicken anti-GFPantibody (Aves Laboratories) recognizes the split-GFP1-10 and thereconstituted GFP protein, while the mouse anti-GFP antibody (G6539,Sigma-Aldrich) recognizes only the reconstituted GFP. Therefore, themouse anti-GFP antibody was used to measure the GRASP signal (anti-GRASP; 1:100) and the polyclonal chicken anti-GFP antibody was used asan internal control for normalizing the GRASP signal. The fluorescenceimages were acquired to minimum signal saturation for quantitation. Themean fluorescence intensities of anti-GRASP and anti-split-GFP1-10 fromeach hemi-neuropil segment (segments 4, 5 and 6) were measured from theconfocal images.

Assessment of dendrite development in nociceptorsThemembrane GFP, mCD8::GFP, was specifically expressed in nociceptorsusing ppk-GAL4 in a wild-type control (wt) and dYPEL3 frameshift mutants(dYPEL3T1-8). Total length of dendrites was measured from the male larvaeof wt and dYPEL3T1-8 using the Simple neurite tracer plugin (Longair et al.,2011) in ImageJ software. Sholl analysis was conducted using the Shollanalysis plugin in ImageJ software (Ferreira et al., 2014).

Analysis of presynaptic arbors of single nociceptorsThe flip-out (Wong et al., 2002) experiment was performed to visualize theterminal axon arbors of single nociceptors. A flip-out cassette (FRT-rCD2-stop-FRT-CD8::GFP) and a heat-shock inducible Flippase (FLP) wasintroduced either in a wild-type control (w1118) or in dYPEL3T1-8 mutantsalong with ppk-GAL4. The 3-day-old larvae grown in grape-agar plate wereheat shocked for 15 min in a 37°C water bath and allowed one more dayof growth at 25°C before being dissected and processed for

immunostaining and imaging. The total presynaptic arbor length wasmanually measured using ImageJ software. Branches shorter than 5 µmwere excluded from the analysis.

Experimental design and statistical analysisAll statistical analysis was performed two-tailed using Prism version 7.04(GraphPad Software). The Chi-square with Fisher’s exact test was used fornociceptive rolling behavior. The Mann–Whitney test was used for calciumimaging (GCaMP) and GRASP experiments. Unpaired Student’s test wasused for presynaptic arbor size and dendritic development analysis. Two-way ANOVA was used for Sholl analysis. P<0.05 was consideredstatistically significant.

AcknowledgementsWe thank Heewon Lee and Lily Lou for technical assistance, Dr Adam Claridge-Chang for sharing the UAS-GtACR1 transgenic flies and Dr Cynthia Tifft for helpfuldiscussions.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: J.H.K., B.Y.; Methodology: J.H.K., N.Z.; Validation: J.H.K., M.S.,G.P., A.L., N.Z.; Formal analysis: J.H.K., M.S., G.P., A.L., N.Z., B.B.; Investigation:J.H.K., M.S., G.P., A.L., N.Z., B.B.; Resources: J.H.K.; Data curation: J.H.K.; Writing- original draft: J.H.K.; Writing - review & editing: J.H.K., M.S., B.Y.; Visualization:J.H.K.; Supervision: J.H.K., B.Y.; Project administration: J.H.K.; Funding acquisition:J.H.K., B.Y.

FundingResearch reported in this study used the Cellular andMolecular Imaging Core facilityat the University of Nevada Reno, which was supported by the National Institute ofGeneral Medical Sciences of the National Institutes of Health (NIH) under grantnumber P20 GM103650, and was supported by the Nevada INBRE (P20GM103440 to J.H.K.) and NIH (R21GM114529 and R01NS104299 to B.Y.).

Supplementary informationSupplementary information available online athttp://dmm.biologists.org/lookup/doi/10.1242/dmm.042390.supplemental

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