INTRODUCTIONEpilepsy is a chronic condition of unpredictable and recurrentseizures produced by abnormal neuronal activity in the centralnervous system (CNS). The etiology of epilepsy is complex, yet hasa strong genetic component. A variety of genes, including thosethat regulate neuronal differentiation, morphology, excitability andsignaling, as well as genes of unknown function, have beenassociated with epilepsy (Wang et al., 2010b). Elucidating thefunctions of these genes would provide a better understanding ofthe disease cause and could potentially contribute to clinicaltherapy.

A genetic screen in epilepsy patients identified mutations in thePRICKLE locus (PK1 and PK2), suggesting an association of PKwith epilepsy (Bassuk et al., 2008; Tao et al., 2011). PK is a corecomponent of the planar cell polarity (PCP) pathway that regulatescell movement and tissue polarity. Loss of pk in Drosophila leadsto disruption of epithelial planar cell polarity in the wing andommatidia (Tree et al., 2002; Jenny et al., 2003; Jenny et al., 2005).Pk mutant mice showed decreased seizure threshold following bothelectrical and convulsant drug induction (Tao et al., 2011).Drosophila pk mutants (spinylegs, sple) likewise display seizure-like

behaviors and, interestingly, the seizure-like behaviors can besuppressed by the anti-epileptic drug valproic acid (Tao et al., 2011).The genetic causes of epilepsy have been linked largely to genesencoding for ion channels or neurotransmitter receptors (Mulleyet al., 2003; Poduri and Lowenstein, 2011). Understanding how PKdysfunction relates to seizures could elucidate previously unknownmechanisms underlying epilepsy.

In vertebrates and chordates, pk modulates directed cellmovement during early embryonic development. At gastrulationstages, sheets of cells undergo convergence extension (CE)movements to form the anterior-posterior axis (A-P axis), andmanipulating pk results in CE defects and shorter A-P axis(Carreira-Barbosa et al., 2003; Takeuchi et al., 2003; Veeman et al.,2003; Jiang et al., 2005). Apart from the role in polarityestablishment, PCP proteins also regulate axon outgrowth andneuronal migration (Wada et al., 2006; Goodrich, 2008; Zhou etal., 2008; Tissir and Goffinet, 2010; Ng, 2012). Drosophila pkpromotes sensory axon outgrowth, suggesting a role as a directionalcue for axons (Mrkusich et al., 2011). pk function is required forthe migration of facial branchiomotor neurons in zebrafish(Carreira-Barbosa et al., 2003; Bingham et al., 2010; Mapp et al.,2010; Mapp et al., 2011). Although patients with PK mutationspresent with a seizure disorder, they do not display severedevelopmental defects and have normal brain MRIs (Bassuk et al.,2008; Tao et al., 2011). These observations suggest that PK1mutations support general CNS development but could lead tosubtle abnormalities in neuronal migration or outgrowth,disrupting signal processing.

Pk1a protein contains PET (Prickle Espinas Testin) and LIM (Lin-1, Isl-1 and Mec-3) protein-protein interaction domains. PK1mutations identified in human epilepsy patients are point mutationsthat lead to single amino acid changes (Bassuk et al., 2008; Tao et

Disease Models & Mechanisms 679

Disease Models & Mechanisms 6, 679-688 (2013) doi:10.1242/dmm.010793

1Department of Biology, University of Iowa, Iowa City, IA 52242, USA2Department of Pediatrics, The University of Iowa Carver College of Medicine, IowaCity, IA 52242, USA*Author for correspondence (diane-slusarski@uiowa.edu)

Received 31 August 2012; Accepted 9 January 2013

© 2013. Published by The Company of Biologists LtdThis is an Open Access article distributed under the terms of the Creative Commons AttributionNon-Commercial Share Alike License (http://creativecommons.org/licenses/by-nc-sa/3.0), whichpermits unrestricted non-commercial use, distribution and reproduction in any medium providedthat the original work is properly cited and all further distributions of the work or adaptation aresubject to the same Creative Commons License terms.

SUMMARY

Epilepsy is a complex neurological disorder characterized by unprovoked seizures. The etiology is heterogeneous with both genetic andenvironmental causes. Genes that regulate neurotransmitters and ion channels in the central nervous system have been associated with epilepsy.However, a recent screening in human epilepsy patients identified mutations in the PRICKLE1 (PK1) locus, highlighting a potentially novel mechanismunderlying seizures. PK1 is a core component of the planar cell polarity network that regulates tissue polarity. Zebrafish studies have shown thatPk1 coordinates cell movement, neuronal migration and axonal outgrowth during embryonic development. Yet how dysfunction of Pk1 relates toepilepsy is unknown. To address the mechanism underlying epileptogenesis, we used zebrafish to characterize Pk1a function and epilepsy-relatedmutant forms. We show that knockdown of pk1a activity sensitizes zebrafish larva to a convulsant drug. To model defects in the central nervoussystem, we used the retina and found that pk1a knockdown induces neurite outgrowth defects; yet visual function is maintained. Furthermore, wecharacterized the functional and biochemical properties of the PK1 mutant forms identified in human patients. Functional analyses demonstratethat the wild-type Pk1a partially suppresses the gene knockdown retinal defects but not the mutant forms. Biochemical analysis reveals increasedubiquitylation of one mutant form and decreased translational efficiency of another mutant form compared with the wild-type Pk1a. Taken together,our results indicate that mutation of human PK1 could lead to defects in neurodevelopment and signal processing, providing insight into seizurepredisposition in these patients.

Mechanisms of prickle1a function in zebrafish epilepsyand retinal neurogenesisXue Mei1, Shu Wu2, Alexander G. Bassuk2 and Diane C. Slusarski1,*

RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

al., 2011). These changes are found across the entire proteininstead of clustering within a specific domain. However, how thesemutants differ from the wild type is not fully understood. Inprevious studies, we have overexpressed mutant Pk1a and Pk2forms in zebrafish to evaluate their pathogenicity. Pk1 and Pk2overexpression induces CE defects and intracellular calcium release(Veeman et al., 2003; Tao et al., 2011) and the mutants showedaltered activities compared with wild-type Pk (Bassuk et al., 2008;Tao et al., 2011). A similar approach has been used for rare Pkmissense mutations associated with neural tube defects (Bosoi etal., 2011). Whereas the overexpression assays have revealedfunctional differences, biochemical changes in the mutant formshave not been evaluated. Moreover, a partial-knockdown contextmight better resemble the physiological state in human patientswith heterozygous mutations. Thus, we set out to investigate thenecessary role of zebrafish Pk1a in neural development and tocharacterize the biochemical properties of the mutant forms.

Zebrafish (Danio rerio) is an emerging model for epilepsy byvirtue of its stereotypic behaviors and response to seizure-inducing

drugs (Hortopan et al., 2010a; Teng et al., 2010; Teng et al., 2011).Seizure-like behaviors in zebrafish larvae and adults have beencharacterized by tracking swimming patterns and examining brainelectrophysiology (Baraban et al., 2005; Hortopan et al., 2010b).Zebrafish is also amenable to genetic manipulations and to imagingtechniques for studying neural development. Here, we use thezebrafish larva as a model and characterize pk1a function in drug-induced seizures. In order to interrogate the role of abnormal pk1aforms in the developing nervous system, we also explore novelaspects of pk1a function in neurite outgrowth in the retina andevaluate biochemical properties of epilepsy-related mutant forms.We confirm the role of pk1a in the sensitivity to drug-inducedseizures; show a requirement of pk1a in retina inner plexiform layer(IPL) organization; and find differential processing of wild-type andmutant forms. These results provide insights into the mechanismsby which mutant forms of PK1 might lead to epilepsy.

RESULTSpk1a knockdown sensitizes zebrafish to PTZ treatmentPentylenetetrazole (PTZ) is a convulsant drug that induces generalmotor seizures in human and mouse (Velisek et al., 1992). PTZalso induces seizure-like behaviors, characterized by whirl-like andjerky swimming patterns, as well as electrophysiological changesin adult and larval zebrafish (Baraban et al., 2005; Hortopan et al.,2010b). Knockdown of known epileptogenic genes such as lgi1(leucine-rich, glioma inactivated 1) in zebrafish leads to sensitizedmotility in response to PTZ (Teng et al., 2010; Teng et al., 2011).We tracked and quantified total swimming distance in response toexposure to the seizure-inducing drug PTZ (Fig. 1). We monitoredthe motility of individual larva in a 48-well culture dish by recording0.5 hours before and 0.5 hours after addition of PTZ to the medium(Fig. 1B). Movement detection software identified ‘small movement’(0.1-10 cm/second) and ‘large movement’ (above 10cm/second).White areas in Fig 1B are regions of little or no movement (below0.1 cm/second). The total activity of an individual larva can thenbe traced within its well. Typically, at 72 hours post fertilization(hpf), wild-type zebrafish displayed small movements around thewell (Fig.  1B, top) (supplementary material Movie 1). Uponexposure to PTZ, wild-type zebrafish showed a marked increasein the amount of movement within the well (Fig.  1B, bottom)(supplementary material Movie 2). The total distance travelledduring the time course (0.5 hours) can be calculated for eachindividual larva and then averaged for the entire plate (48 wells).We found increases in total activity associated with increasing dosesof PTZ (Fig. 1C).

Pk mutant mice show increased sensitivity to PTZ exposure. Todetermine whether reduced activity of pk1a sensitizes zebrafish toPTZ treatment, we utilized gene knockdown and PTZ treatmentstrategies. In order to evaluate the entire range of response, weselected a PTZ dose that induces a mild yet statistically significantincrease in activity in wild-type zebrafish (5 mM; Fig. 1C, Fig. 2D).Because pk1a knockdown induces A-P axis defects (Carreira-Barbosa et al., 2003; Veeman et al., 2003), with shorter or curvedtrunks that would compromise swimming (Fig.  2C), we nextidentified a sub-phenotypic dose of antisense morpholino-oligonucleotide (MO) directed against pk1a that generates straight-bodied zebrafish to be used for the motility assays (Fig. 2B). Wefound that the straight-bodied pk1a-MO-injected larvae

dmm.biologists.org680

PCP in epilepsy and retina neurogenesisRESEARCH ARTICLE

TRANSLATIONAL IMPACT

Clinical issueEpilepsy is a chronic condition characterized by recurrent, spontaneousseizures. Although epilepsy can be caused by non-genetic factors includingstroke, traumatic brain injury and brain infection, genetic causes make asignificant contribution to epilepsy development. Mutations in genes thatregulate ion channels and neurotransmitters are shown to be frequentlyinvolved in epileptogenesis. Understanding the underlying genetics andpathophysiology of epilepsy is necessary to inform therapies that could reduceor offset the incidence of seizures.

ResultsMutations at the human PRICKLE (PK) locus are associated with epilepsy. Toinvestigate the mechanistic role of PK in epilepsy, the authors characterizedthe functions of wild-type and epilepsy-related mutants in a zebrafish model.Using a zebrafish larva motility assay, the authors showed that the convulsantdrug PTZ induces hyperactivity in larva swimming; however, larva with pk1agene knockdown were found to be more sensitive to PTZ compared with thecontrol. The protein encoded by the PK1a gene, PK1a, is a planar cell polarityprotein that regulates tissue polarity and directed cell movements and isinvolved in neuronal migration and outgrowth. To probe the role of PK1a inneural development, the authors examined neuronal outgrowth in a zebrafishretinal model. Their analysis revealed a necessary role for PK1a in neuriteorganization in the retina inner plexiform layer. Because the mutationsidentified in human patients are not clustered in a single domain but aredistributed across the protein, the authors tested the different mutant formsfor loss of function. Although wild-type PK1a overexpression in the zebrafishembryos was able to partially suppress the neuronal outgrowth defect, mutantforms were found to be less active. In addition, biochemical analysis showedthat one of the mutant forms of PK1a, R150H, was more strongly ubiquitylatedthan the wild-type or Y465H mutant protein.

Implications and future directionsAn association of PK with epilepsy has been described in fly and mousemodels, but the underlying mechanisms are not well understood. Here, theauthors demonstrated the utility of a zebrafish larva epilepsy model andidentified novel functions of PK in retinal neurogenesis. Mutations in PK mightcontribute to epilepsy via its necessary roles in neuronal outgrowth duringearly development. Patients expressing mutant forms that retain partialfunction can be predisposed to seizures. Future utilization of the motility assayand retina model could enable epilepsy-drug screening.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

(morphants) were similar to control morphants in total movement(Fig.  2D, before PTZ). By contrast, after exposure to PTZ, pk1amorphants showed a significantly higher level of activity comparedwith control morphants (Fig. 2D, after PTZ). To confirm that thesensitivity to PTZ in pk1a morphants is mediated throughmechanisms that promote seizures, we tested the ability of theantiepileptic drug valproic acid (VPA) to suppress the increasedmotility. After pretreatment with VPA for 1 hour before addingPTZ, there was no statistically significant difference betweencontrol and pk1a morphants (supplementary material Fig. S1).Taken together, we conclude that pk1a knockdown sensitizes thezebrafish to the seizure-inducing dug PTZ.

pk1a knockdown induces neurite outgrowth in the retinapk has been shown to regulate the migration of facialbranchiomotor neurons in the hindbrain (Carreira-Barbosa et al.,2003; Mapp et al., 2010; Mapp et al., 2011; Mrkusich et al., 2011;Sanchez-Alvarez et al., 2011), but little is known about the affectsof pk1a dysfunction in the central nervous system (CNS). Giventhe PTZ-sensitivity in pk1a knockdown embryos, we sought amodel to explore mechanisms of pk1a function in CNSdevelopment. Because the retina originates as an outgrowth of thebrain, it serves as a model for the CNS. Whole mount in situhybridization of the 3 dpf larval brain shows pk1a expressionthroughout the brain, with enriched expression in the eye(Fig. 3A,B; supplementary material Fig. S2). The retina is organized

in layers with stereotypical cell types and dendritic projectionssimilar to but less complicated than the CNS. We furthercharacterized the expression of pk1a transcript within the eye andfind it enriched in the retinal ganglion cell layer (RGC), innernuclear layer (INL) and the lens at 3 dpf (Fig. 3C,D). pk1a is alsoexpressed in adult retina as determined by RT-PCR (data notshown). To determine whether pk1a is necessary for globalpatterning of the retina, we performed histological analysis of pk1amorphants by hematoxylin and eosin (H&E) staining. At 3 dpf, pk1amorphants have fully laminated retinas similar to wild-type andcontrol morphants (Fig. 3E-G). The photoreceptor outer segmentsin the central retina revealed no overt differences compared withwild-type and control morphants (Fig. 3E�-G�). However, the inner

Disease Models & Mechanisms 681

PCP in epilepsy and retina neurogenesis RESEARCH ARTICLE

Fig. 1. PTZ induces hyperactivity in swimming behavior in a dose-dependent manner. (A)Flow chart of the motility assay and analysis.(B)Representative pictures generated in the Viewpoint Software showing thetrack of a larva before and after PTZ treatment; total movement over30 minutes. Red circles depict the shape of the well; green and red lines markthe small and large movements, respectively. (C)Graph of total half-houractivity of larva treated with different doses of PTZ. Data presented as mean + s.e.m.

Fig. 2. pk1a knockdown sensitizes zebrafish to PTZ treatment.(A-C)Morphology of wild-type and pk1a-MO-injected embryos at 2 dpf: wild-type uninjected embryo (A); pk1a-MO-injected embryos ranging from‘normal-like’ (B) to curved body axis (C). (D)Graph of total half-hour activitybefore and after PTZ treatment for larvae injected with control-MO and pk1a-MO. Wilcoxon Rank Sum test found no significant increase (ns) in activity ofcontrol-MO and pk1a-MO injected larvae prior to PTZ treatment. There was asignificant increase in activity in control after PTZ compared with controlbefore PTZ (P

segments (Fig.  3E�-G�), in particular the IPL, displayed modestdisorganization sometimes with nuclei found within the IPL(Fig. 3G�, arrowhead).

The IPL is a synaptic layer that bears connections between cellsin the ganglion cell layer and inner nuclear layer. To more deeplyinterrogate the subtle anomalies in IPL histology, we used thetransgenic line HuC:gfp that expresses GFP in a subset of neuronalcells, the ganglion cells and amacrine cells in the inner nuclear layer(Link et al., 2000; Park et al., 2000). Both cell types extend theirprojections into the IPL, forming stratified arborizations thatpresent as two continuous tracks in the HuC:gfp line. MO-injectedHuC:gfp transgenic embryos were fixed at 3 dpf and transverse

sections from the central retina collected for confocal imaging(Fig. 4A-H). In wild-type and control morphants, synapticconnections in the IPL were readily visible as two stratified tracks(Fig. 4A�,B�). In pk1a morphants, we observed defects in the trackorganization. About 41% of the pk1a morphants displayed acomplete loss of parallel organization of the two stratified tracksand were classified as severe (Fig.  4C�). An additional 14% hadregions of discontinuous or disorganized parallel tracks, classifiedas mild (Fig. 4D�, arrowheads). To verify the specificity of the pk1aknockdown defect, we utilized a second MO directed against pk1awith different target sequences. The second pk1a-MO generatedan IPL defect with a penetrance of 69% (all defects were severe)(Fig. 4E,E�).

In the early embryo, mis-expression of pk1a generates similardefects to that of knockdown (Carreira-Barbosa et al., 2003;Veeman et al., 2003; Bassuk et al., 2008). We found that mis-expression of pk1a RNA in wild-type embryos also disrupted IPLorganization (Fig. 4F,G). In order to test for suppression of the MO-induced IPL defects, we identified the dose of pk1a RNA that doesnot induce an overexpression defect (Fig.  4F,F�). We used a lowdose (40 pg) of pk1a RNA that does not lead to an overexpressiondefect in the IPL (Fig. 4F,F�). Co-injection of pk1a-MO with thisdose of RNA partially suppressed the MO-induced IPL defect(Fig.  4H,H�). We conclude that pk1a function is required forappropriate arborization within the IPL and that this defect is, inpart, suppressed by wild-type Pk1a.

Human epilepsy patients with PK1 mutation have normal visualperception (Bassuk et al., 2008; Tao et al., 2011), yet we observeretinal defects in pk1a morphants. We next evaluated visualfunction of pk1a morphants. The vision startle response in zebrafishis a natural escape response that is elicited when embryos areexposed to rapid changes in light intensity (Baye et al., 2011).Visually responsive 5 dpf embryos changed their swimmingbehavior when there was a short block in a bright light source(supplementary material Fig. S3A-C). The assay was repeated fivetimes and the average number of responses reported. Wild-typeembryos responded an average of 3.6 times. By contrast, the visuallyimpaired cone-rod homeobox (crx) morphants responded only 2.2times (supplementary material Fig. S3D) (Baye et al., 2011). pk1amorphants with a straight body axis showed a response profilesimilar to the wild type (supplementary material Fig. S3D). Theseobservations suggest that pk1a knockdown does not compromisebasic visual function and indicate that the IPL defect might impacthigher order processing. The extent to which similar processingdefects, if present in the CNS, relate to epilepsy is unknown.

Pk1a epilepsy mutant forms display distinct functional andbiochemical properties compared with the wild-type formMutations in PK1 lead to single amino acid changes that locatein different parts of the protein, and PK1 patients have normalstature. Given that the mouse Pk1-null mutant is homozygouslethal and that pk1a complete knockdown in zebrafish leads totrunk defects, we reasoned that each of the mutations found inhumans reflects a partially functional product. Moreover, distinctdomains harboring mutations might compromise specific aspectsof Pk1 function. To test this possibility, we performed functionalanalyses and investigated the biochemical properties of themutant forms.

dmm.biologists.org682

PCP in epilepsy and retina neurogenesisRESEARCH ARTICLE

Fig. 3. pk1a is expressed in the brain and retina and is not required foroverall retinal lamination. (A,B)Whole-mount in situ hybridization of 3 dpfembryos. Images are from the dorsal view. An antisense RNA riboprobe is usedto determine the expression pattern of pk1a, and a riboprobe from the sensestrand is used as a control. (C,D)Sections of the eye after whole-mount in situhybridization at 3 dpf: sense control (C) and pk1a antisense (D). Arrows showdifferent layers in the retina. (E-G�) H&E staining of eye sections at 76-78 hpf.(E-G)Whole retina sections of uninjected (E), control-MO-injected (F) andpk1a-MO-injected larvae (G); magnification 40×. (E�-G�) Enlarged areas of thewhite boxes in E-G; the arrowhead in G” indicates nuclei disorganization;magnification 63×. PR, photo-receptors; INL, inner nuclear layer; IPL, innerplexiform layer; RGCs, retina ganglion cells; OPL, outer plexiform layer; ONL,outer nuclear layer. Scale bars: 5 μm.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

We generated and expressed zebrafish Pk1a carrying analogousmutations. The R150H mutation is an amino acid change ofarginine 150 to histidine in the first LIM domain and the Y465Hmutation is a change of tyrosine 465 to histidine in the middle ofthe protein (Fig. 5A) (Tao et al., 2011). In overexpression assays,we monitored IPL formation and found that excess R150H andY465H pk1a RNA both led to IPL defects (supplementary materialFig. S4). To evaluate activity under more physiologically relevantconditions, we tested the extent to which the mutant RNA cansuppress the MO-induced IPL defect. As shown in Fig.  4, pk1amorphants had mild and severe IPL defects and were statisticallydifferent to control embryos (Fig. 5B). Co-injection of wild-typepk1a RNA with pk1a-MO not only reduced the severity of the IPLdefect but also the embryos were no longer statistically significantlydifferent to uninjected controls (Fig. 5B). By contrast, co-expressionof Y465H led to a larger percentage of severe defects (Fig. 5B). Co-expression of R150H with pk1a-MO demonstrated a shift fromsevere to mild IPL defects but total defects were still significantlydifferent from uninjected control (Fig. 5B). Thus, wild-type pk1aRNA can suppress the MO-induced defect. Although the R150Hmutation seemed to attenuate the MO-induced defect, the effectwas not statistically significant. The mutant form Y465H, on theother hand, seemed to accentuate the MO-induced IPL defect. Wenext explored biochemical changes that could account for thedifferent functional activities.

We first determined expression of the myc-tagged mutant formsusing western blot analysis of embryo lysates. At 80% epiboly (~8hpf), we found reduced expression of the R150H form comparedwith the wild-type Pk1a. By contrast, Y465H was expressed at thesame levels as the wild-type protein, if not slightly higher (Fig. 6A).Although wild-type and mutant proteins were not detectable at 76hpf, the stage at which we evaluate the IPL formation (data notshown), we did find expression of all the forms at 30 hpf when initialretinal ganglion cells start to grow processes (supplementarymaterial Fig. S5A) (Schmitt and Dowling, 1996). The reducedexpression of the R150H form of Pk1a could be due to increasedturnover or less efficient translation than the wild-type form. Totest the possibility of turnover, we expressed myc-tagged R150HPk1a and treated the embryos with the proteosomal inhibitor MG-132. The expression level of R150H Pk1a was unchanged followingdrug treatment (supplementary material Fig. S5B), suggesting thatthe lower R150H expression levels might be a result of lowtranslational efficiency.

Pk1 was shown to be ubiquitylated in cell culture (Narimatsu etal., 2009). To determine whether ubiquitylation is altered in themutant forms, we co-injected HA epitope-tagged ubiquitin withmyc-tagged Pk1a forms. Denatured embryo lysates wereimmunoprecipitated with anti-myc antibody-conjugated agarosebeads and probed with an antibody specific for the HA epitope bywestern blot. The epitope-tagged ubiqutin was readily incorporated

Disease Models & Mechanisms 683

PCP in epilepsy and retina neurogenesis RESEARCH ARTICLE

Fig. 4. pk1a knockdown induces inner plexiform layer defects.(A-H�) Sections at 76-78 hpf in the HuC:gfp transgenic line. Greenchannel, GFP; blue channel, To-Pro3 nuclear counter-staining. (A-H)Whole eye visualized at 63×. Representative sections fromuninjected embryos (A) and from embryos injected with control-MO(B); pk1a-MO, showing severe IPL defects (C); pk1a-MO, showing mildIPL defects (D); a second pk1a-MO (E); 40 pg pk1a RNA (F); 80 pg pk1aRNA (G); pk1a-MO plus 40 pg pk1a RNA (H). (A�-H�) Digital 3× zoom ofthe white boxed areas in A-H, showing the green channel only. In theuninjected (A�) and control-MO-injected embryos (B�), there are twoclearly stratified tracks. pk1a morphants exhibit either loss of theorganization (C�) or discontinuous tracks (arrowheads in D�). A secondpk1a-MO also induced similar IPL defects (E�). Although a low dose ofpk1a RNA injection does not show a defect (F�), a higher dose does(G�). The defects can be partially suppressed by a low dose pk1a RNAco-expression (H�). Scale bar: 10 μm.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

into zebrafish proteins (Fig. 6B, input: HA). To insure equal loadingof Pk1a mutant forms, we adjusted the pull-down samplesaccording to their abundance in myc-tagged proteins (Fig. 6B, IB:myc). We then proportionally loaded pull-down samples for anti-HA immunoblot. Interestingly, we found that the Y465H mutantPk1a was more strongly ubiquitylated than the wild type or R150Hform (Fig.  6B, IB: HA). These results further confirm that thedifferential expression level of the R150H mutant occurs withoutdetectable ubiquitylation and is probably independent ofproteosomal degradation. Moreover, the increased ubiquitylationin Y465H Pk1a appears unrelated to degradation and could impactlocalization or protein-protein interactions. These data suggest thatthe distinct biochemical properties displayed by wild-type andmutant forms of Pk1a reflect the functional diversity of the differentmutant forms in human patients.

DISCUSSIONHere, we describe the development of a zebrafish model to elucidatethe mechanisms by which PK1 dysfunction leads to epilepsy. Wefirst demonstrate that pk1a knockdown sensitizes zebrafish larvaeto the convulsant drug PTZ. This finding is consistent with whatwas reported for Pk mutant mice and flies (Tao et al., 2011). Wefind broad pk1a expression in the larva brain with enrichedexpression in the retina. Consistently, pk1a knockdown leads toneurite outgrowth defects in the retina, similar to defects observedin the developing fly nervous system (Tao et al., 2011). These results

further highlight the evolutionary role of Pk in regulating seizuresand neurodevelopment. Furthermore, we find that mutant formsof Pk1 associated with human epilepsy display distinct functionaland biochemical properties.

Human patients heterozygous for the R150H and Y465Hmutations of PK1 are otherwise developmentally normal. The factthat Pk1 knockout in mice is lethal suggests that the Pk1 gene iscrucial for embryonic development. In the case of human patientswith PK1 mutations, it is possible that either PK2 can compensatefor PK1 function or these mutations are hypomorphic, maintaininga partially functional PK1 product, or both. Pk is a core PCP protein,and the amount of product as well as the asymmetrical localizationof core PCP proteins is necessary for correct cell polarization (Treeet al., 2002; Jenny et al., 2003; Veeman et al., 2003). Therefore, eithertoo much or too little of the protein would lead to similarphenotypes. The extent to which the IPL defect is a PCP phenotyperemains to be determined; however, reduced Wnt5b function (aWnt that generates PCP outputs) leads to defects in IPL formation(Lin et al., 2010). Moreover, we demonstrate that Pk1aoverexpression leads to an IPL defect as the mutant forms do, whichis consistent with the notion that the mutant forms retain partialor aberrant activity. Although the mutant forms of Pk1ademonstrate overexpression phenotypes, our utilization of the pk1aknockdown approach with expression of the wild-type and mutantforms revealed functional differences in the ability to suppress theIPL defect. This approach might reflect the physiology of the humanpatients with heterozygous mutations and could provide insightsinto the development of the CNS and the mechanisms underlyingthe epilepsy phenotype in PK mutants.

Our biochemical analyses indicate that specific mutationsaffect distinct aspects of pk1a protein function that, whencompromised, display overlapping phenotypes. The reducedexpression of R150H Pk1a, even after treatment with theproteosome inhibitor MG-132, suggests that this form istranslated less efficiently than wild-type Pk1a. Similartranslational efficiency could lead to reduced PK1 protein inhuman patients and thereby contribute to the epileptic phenotype.The R150H mutant form attenuates the MO-induced defect butnot to significant levels. This suggests that the protein, whenprovided in exogenous levels, can function like the wild type.

The Pk1 protein undergoes post-translational modification. Incontrast to previous evidence in cell culture (Narimatsu et al., 2009),the wild-type zebrafish pk1a form did not show extensiveubiquitylation. We reason that in the cell culture studies, additionof E3 ubiquitin ligase as well as other Pk1-interacting factorsenhanced this modification whereas, in our studies, the extent ofubiquitylation relies on endogenous ubiquitin ligases andinteracting factors expressed in the early embryo. Despite thedependence upon endogenous factors in the embryo, we find robustubiquitylation of the Y465H mutant form. In addition to proteolyticoutcomes, ubiquitylation also regulates protein-protein interactionsand protein trafficking in endosomal compartments (Scita and DiFiore, 2010; Piper and Lehner, 2011; Dupont et al., 2012).Ubiquitylation has also been proposed to act as a general modulatorof protein function similar to the role of phosphorylation (Dupontet al., 2012). These alternative functions attributed to ubiquitylationmight apply to the Y465H mutant form of Pk1a as it is stablyexpressed in the zebrafish. The ubiquitylated Y465H form might

dmm.biologists.org684

PCP in epilepsy and retina neurogenesisRESEARCH ARTICLE

Fig. 5. pk1a epilepsy mutant forms display reduced ability to suppress theMO-induced IPL defect. (A)Schematic of the zebrafish Pk1a protein. Grayboxes show the PET and LIM protein-protein interaction domains. Arrowsdenote the position of mutations analogous to those found in human epilepsypatients. (B)Graph showing the range of IPL defects in uninjected controls,pk1a morphants and morphants with mis-expressed mutant Pk1a. Treatmentwith pk1a-MO caused an increase in mild and severe defects. Wild-type pk1aRNA was able to increase the percentage of normal retinas and retinas withmild defects and decrease the percentage with severe defects; Y465H failed todo so and R150H seemed to increase the percentage of mild defects. However,neither mutant form suppressed the MO-induced IPL defects as efficiently asthe wild-type form. Light shading represents normal IPL; medium darkshading represents mild defects, and dark shading represents severe defects.*P

be sorted through a different compartment than the wild-type orR150H forms of Pk1a, or have different abilities to interact withbinding partners (Shimojo and Hersh, 2003; Jenny et al., 2005;Shimojo and Hersh, 2006). Our study revealed that the two mutantforms display distinct biochemical properties that could contributeto epileptogenesis by different mechanisms.

Epileptic seizures are thought to arise from the imbalance ofexcitatory and inhibitory signaling in the central nervous system.PTZ generates motor seizures in zebrafish by antagonizing theinhibitory γ-aminobutyric acid (GABA)-mediated post-synapticsignaling (Macdonald and Barker, 1977; Baraban et al., 2005;Hortopan et al., 2010b). Although our findings imply that partialloss of pk1a potentiates seizures in response to PTZ, implicatingthe GABA pathway in pk1 mutant-related epilepsy, themechanism is unclear. One possibility is that pk1a is necessaryfor the developmental refinement of axons and dendrites and thatsubtle deficits in the signal processing within excitatory andinhibitory neurons render the network prone to seizures. Giventhe IPL organization defects we observe in pk1a knockdownretina, it is possible that the connectivity is affected and thusabnormal signal processing could contribute to the CNS responseto stimuli. Similarly, mutations have been shown to link toepilepsy in genes that regulate neuronal proliferation, migrationand circuit remodeling (reviewed by Bozzi et al., 2012). For

example, these genes include Reln, which codes for Reelin and isresponsible for the migration of cortical neurons (D’Arcangeloand Curran, 1998; D’Arcangelo, 2006; Patrylo et al., 2006); Dlx,which codes for transcription factors that are necessary forcortical interneuron differentiation and migration (Anderson etal., 1997; Cobos et al., 2005; Wang et al., 2010a); and Lgi1, lackof which cause defects in pruning and maturation of hippocampalglutamatergic neurons (Kalachikov et al., 2002; Striano et al.,2011). Interestingly, the Dlx5/6+/− mice display electrographicspontaneous seizures without any gross histological abnormalitiesin the cortex (Wang et al., 2010a), suggesting a connectivity issue.Whether similar types of defects exist in the CNS of pk1aknockdown zebrafish is not known. Moreover, it is not clearwhether human PK patients have subtle retinal axonalorganization defects.

In summary, we describe the use of zebrafish larvae as a modelto study epilepsy mechanisms. We provide evidence that reducedpk1a function leads to increased sensitivity to PTZ-induced seizurebehaviors. We also demonstrate the role of pk1a in the developingretina, potentially also in CNS development, which suggests thatsynaptic connectivity might affect signal processing. Biochemicalcharacterization of mutant forms could inform treatmentparadigms in human patients. Further studies to dissect otherepilepsy mechanisms with the zebrafish model will be useful forilluminating potential treatment paradigms for human patientsharboring specific mutations.

MATERIALS AND METHODSEthical statementAll the work with zebrafish was approved by University AnimalCare and Use Committee of the University of Iowa.

Zebrafish maintenanceZebrafish adults were maintained under standard conditions andeggs collected from natural spawnings (Westerfield, 1993). Stagingof the embryos was performed according to Kimmel et al. (Kimmelet al., 1995).

Morpholinos, expression constructs and primersControl MO, 5�-CCTCTTACCTCAGTTACAATTTATA-3�; pk1a-MO, 5�-CAGCTCCATCACTAACACCCCCTCA-3� and pk1asecond MO, 5�-GCCCACCGTGATTCTCCAGCTCCAT-3� werepurchased from Gene Tools. The second pk1a-MO completelyoverlaps with pk1a RNA sequence so was not used for rescuestudies. HA-Ub expression construct was a gift from DouglasHouston (University of Iowa, Iowa City, IA). The myc-pk1a wild-type form was cloned from 8-12 somite stage cDNA library withGeneAmp highfidelity polymerase (Applied Biosystems, LifeTechnologies, Carlsbad, CA). PCR primers for cloning pk1a were:(forward) 5�-ATGGAGCTGGAGAATCACGGTGG-3� and(reverse) 5�-AATTCCCTCTCAAAGTGGGC-3�. PCR productswere cloned into PCR8/GW/TOPO entry vector and recombinedwith the destination vector with an N-terminal 6× myc (fromNathan Lawson’s Lab at University of Massachusetts MedicalSchool, Worcester, MA). TA cloning and LR recombination kitswere purchased from Life Technologies (Carlsbad, CA). The myc-pk1a mutant forms were generated by site-directed mutagenesisas described (Tao et al., 2011). In vitro transcription was performed

Disease Models & Mechanisms 685

PCP in epilepsy and retina neurogenesis RESEARCH ARTICLE

Fig. 6. pk1a epilepsy mutants display distinct biochemical properties.(A)Western blot of myc-tagged wild-type (wtPk1) and mutant forms of Pk1a.Protein lysates were made at 80% epiboly stage. β-actin was used as a loadingcontrol. (B)Pull-down assays showing the extent to which wild-type (wt) andmutant forms are ubiquitylated. HA-Ub and pk1a RNAs were co-injected intoone-cell stage embryos and at 80% epiboly total cell lysates were pulled downby anti-myc antibody beads and blotted for anti-myc or anti-HA. Embryosinjected with HA-Ub without pk1a RNA were subjected to anti-myc pull-downas a negative control.

Dise

ase

Mod

els &

Mec

hani

sms

D

MM

to make synthetic RNAs using the mMessage mMachine in vitrotranscription kit (Ambion, Life Technologies, Carlsbad, CA).Microinjections were done at the 1-2 cell stage by injecting intothe yolk. Needles for injections were calibrated after each injectionset. The injection volume varied between 2.0 nl/pump and 3.0nl/pump. For the motility assay and the retina axon outgrowthanalyses, 1.0 ng and 2.0-3.0 ng pk1a-MO was injected into theembryos, respectively. For the vision assay, 1.0 ng crx-MO was used.RNA injection doses were 40 pg and 80 pg wild-type pk1a for rescueexperiments and 250 pg wild-type and mutant forms forbiochemical analyses.

Drug treatment and motility assayAge-matched zebrafish larvae were incubated at 28.5°C. Larvae at60 hpf were transferred individually into a 48-well culture dish andincubated in the Zebrabox overnight. Each well contained 1.2 mlegg water. Larvae at 3 dpf were monitored for swimming behaviorby the Zebrabox and the Viewpoint Software (Viewpoint LifeSciences, Lyon, France). The behavior was recorded once everysecond for 0.5 hours before and after PTZ treatment.Detection/movement threshold was set at 0.1/10. Activities below0.1, from 0.1 to 10, and above 10 were designated by the softwareas ‘freezing’ (white areas in Fig. 1A), ‘small movement’ (green linesin Fig. 1A) and ‘large movement’ (red lines in Fig. 1A), respectively.For quantification, total swimming distances across the whole 0.5hours, including small and large movements, were calculated andaveraged by larvae number. Given that the total activity byindividual larvae was not normally distributed (by the Shapiro-Wilknormality test), the Wilcoxon Rank Sum Test was used.

Powdered PTZ (Sigma-Aldrich, St Louis, MO) was dissolved inegg water to make fresh working stock before each experiment.For dose-dependent response experiments, PTZ working stock wasadded to each well to achieve the desired final concentration in afinal volume of 1.6 ml. For tracking the MO-injected larvae, 0.4 mlof 20 mM PTZ was added to each well to make a final 5 mMconcentration. To minimize the disturbance to the larvae duringthe addition of PTZ, the room was kept dark and quiet.

Powdered VPA (Sigma-Aldrich) was dissolved in egg water tomake a working stock of 3 mM. Larva were incubated in 0.8 mlegg water overnight and tracked for 1 hour before addition of 0.4ml VPA working stock, making the final VPA concentration 1 mM.Then 0.4 ml of 20 mM PTZ was added to make a final 5 mM finalconcentration and the larva were tracked for 1 hour.

Larvae of 3 dpf in egg water or 15 mM PTZ were placed in anEppendorf tube cap and video-taped at about 7 frames/second.Extracted image sequences from the video were imported intoQuicktime and made into a movie at 10 frames/second.

In situ hybridizationWhole mount in situ hybridization was performed according topreviously described protocols (Thisse et al., 1993). Embryos werefixed at 76-78 hpf in 4% paraformaldehyde in 1× PBS. Degoxigenin-UTP labeled RNA riboprobes were made from linearized constructsusing the MAXIscript in vitro transcription kit (Ambion, LifeTechnologies, Carlsbad, CA). Brain sections (10 μm) were collectedfrom post-fixed embryos by cryosectioning as described below.Sections were mounted in 1:1 sterile glycerol and 1× PBS andimaged at 20× magnification.

Retina histology and fluorescence microscopyMO-injected embryos were fixed at 76-78 hpf for 24 hours andinfiltrated in 15% sucrose and 30% sucrose and in OCT (optimalcutting temperature medium, Sakura, Alphen aan den Rijn, TheNetherlands) overnight at 4°C. Then, embryos were aligned andembedded in OCT, frozen and sectioned at −21°C. Sections (12μm) were mounted on glass slides and left to dry overnight. H&Estaining was performed according to standard protocols. Forfluorescence microscopy, HuC:gfp transgenic embryos were used.Dried slides were rehydrated with 1× PBS and stained with To-Pro3 (Molecular Probes, Life Technologies, Carlsbad, CA) diluted1:1000 in PBS. Sections (12 μm) were mounted with Vectorshieldmounting medium (Vector Laboratories, Burlingame, CA),coverslipped and imaged with Leica TCS SP2 confocal microscopeat 63× magnification with a 3× zoom. Zoomed images weremaximum projections of four consecutive scans in a series.

Western blot and pull-down assayRNA-injected embryos were lysed at 80% epiboly stage and proteinsamples analyzed with mouse anti-myc antibody (9E10; Santa CruzBiotechnology, Santa Cruz, CA) in 1:10,000 dilution. Rabbit anti-β-actin antibody (Sigma-Aldrich) in 1:2000 dilution was used asloading control. For MG-132 treatment, RNA-injected embryoswere dechorionated at the ~1000-cell stage. DMSO or 10 mM MG-132 (EMD Millipore, Billerica, MA) was added to the egg water ata dilution of 1:1000. Embryo lysate was collected at 80% epibolystage. For pull-down assays, myc-tagged proteins wereimmunoprecipitated with anti-myc antibody diluted 1:400 usingprotein A/G agarose beads (Fisher Scientific, Loughborough, UK).The beads were then washed three times with buffer (20 mM Tris,100 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 2% SDS, 100 μMPMSF and 5 μg/ml leupeptin) and boiled at 100°C for 5 minutes.

Vision startle response assayThe vision startle response assay was as previously described(Nishimura et al., 2010; Pretorius et al., 2010; Baye et al., 2011) andwas modified from Easter and Nicola (Easter and Nicola, 1996).Zebrafish larvae at 5 dpf were light-adapted for at least 1 hour. Avisual stimulus was applied to the larvae by rapidly closing andopening the shutter, allowing for ~1 second of darkness. A positiveresponse would be seen as an abrupt direction change of the larvaduring the 1 second (either at the immediate onset of darkness orat the onset of light). Five trials were performed on the same fishspaced 30  seconds apart. A blunt needle probe was used to testthe embryo for touch-responsiveness. Larvae with no touch-response were excluded from the data analysis. Data were plottedas the average number out of five trials that a larva was scored aspositive.ACKNOWLEDGEMENTSWe thank Trudi Westfall and Hilary Griesbach for technical expertise, Lisa Baye andPamela Pretorius for providing reagents and feedback to the manuscript, andRobert A. Cornell and Amanda R. Decker for their help with the behavior assay anduse of the Zebrabox.

COMPETING INTERESTSThe authors declare that they do not have any competing or financial interests.

AUTHOR CONTRIBUTIONSD.C.S. and A.G.B. conceived and designed the experiments. X.M. performed theexperiments. S.W. contributed to the reagents. D.C.S. and X.M. analyzed the data.X.M. and D.C.S. wrote the paper.

dmm.biologists.org686

PCP in epilepsy and retina neurogenesisRESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

FUNDINGThis work is supported by the National Institutes of Health [grant numberR01NS064159 to A.G.B. and D.C.S.].

SUPPLEMENTARY MATERIALSupplementary material for this article is available athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.010793/-/DC1

REFERENCESAnderson, S. A., Eisenstat, D. D., Shi, L. and Rubenstein, J. L. (1997). Interneuron

migration from basal forebrain to neocortex: dependence on Dlx genes. Science 278,474-476.

Baraban, S. C., Taylor, M. R., Castro, P. A. and Baier, H. (2005). Pentylenetetrazoleinduced changes in zebrafish behavior, neural activity and c-fos expression.Neuroscience 131, 759-768.

Bassuk, A. G., Wallace, R. H., Buhr, A., Buller, A. R., Afawi, Z., Shimojo, M., Miyata,S., Chen, S., Gonzalez-Alegre, P., Griesbach, H. L. et al. (2008). A homozygousmutation in human PRICKLE1 causes an autosomal-recessive progressive myoclonusepilepsy-ataxia syndrome. Am. J. Hum. Genet. 83, 572-581.

Baye, L. M., Patrinostro, X., Swaminathan, S., Beck, J. S., Zhang, Y., Stone, E. M.,Sheffield, V. C. and Slusarski, D. C. (2011). The N-terminal region of centrosomalprotein 290 (CEP290) restores vision in a zebrafish model of human blindness. Hum.Mol. Genet. 20, 1467-1477.

Bingham, S. M., Sittaramane, V., Mapp, O., Patil, S., Prince, V. E. andChandrasekhar, A. (2010). Multiple mechanisms mediate motor neuron migrationin the zebrafish hindbrain. Dev. Neurobiol. 70, 87-99.

Bosoi, C. M., Capra, V., Allache, R., Trinh, V. Q.-H., De Marco, P., Merello, E.,Drapeau, P., Bassuk, A. G. and Kibar, Z. (2011). Identification and characterizationof novel rare mutations in the planar cell polarity gene PRICKLE1 in human neuraltube defects. Hum. Mutat. 32, 1371-1375.

Bozzi, Y., Casarosa, S. and Caleo, M. (2012). Epilepsy as a neurodevelopmentaldisorder. Front. Psychiatry 3, 19.

Carreira-Barbosa, F., Concha, M. L., Takeuchi, M., Ueno, N., Wilson, S. W. and Tada,M. (2003). Prickle 1 regulates cell movements during gastrulation and neuronalmigration in zebrafish. Development 130, 4037-4046.

Cobos, I., Calcagnotto, M. E., Vilaythong, A. J., Thwin, M. T., Noebels, J. L.,Baraban, S. C. and Rubenstein, J. L. (2005). Mice lacking Dlx1 show subtype-specific loss of interneurons, reduced inhibition and epilepsy. Nat. Neurosci. 8, 1059-1068.

D’Arcangelo, G. (2006). Reelin mouse mutants as models of cortical developmentdisorders. Epilepsy Behav. 8, 81-90.

D’Arcangelo, G. and Curran, T. (1998). Reeler: new tales on an old mutant mouse.BioEssays 20, 235-244.

Dupont, S., Inui, M. and Newfeld, S. J. (2012). Regulation of TGF-β signaltransduction by mono- and deubiquitylation of Smads. FEBS Lett. 586, 1913-1920.

Easter, S. S., Jr and Nicola, G. N. (1996). The development of vision in the zebrafish(Danio rerio). Dev. Biol. 180, 646-663.

Goodrich, L. V. (2008). The plane facts of PCP in the CNS. Neuron 60, 9-16.Hortopan, G. A., Dinday, M. T. and Baraban, S. C. (2010a). Spontaneous seizures and

altered gene expression in GABA signaling pathways in a mind bomb mutantzebrafish. J. Neurosci. 30, 13718-13728.

Hortopan, G. A., Dinday, M. T. and Baraban, S. C. (2010b). Zebrafish as a model forstudying genetic aspects of epilepsy. Dis. Model. Mech. 3, 144-148.

Jenny, A., Darken, R. S., Wilson, P. A. and Mlodzik, M. (2003). Prickle and Strabismusform a functional complex to generate a correct axis during planar cell polaritysignaling. EMBO J. 22, 4409-4420.

Jenny, A., Reynolds-Kenneally, J., Das, G., Burnett, M. and Mlodzik, M. (2005).Diego and Prickle regulate Frizzled planar cell polarity signalling by competing forDishevelled binding. Nat. Cell Biol. 7, 691-697.

Jiang, D., Munro, E. M. and Smith, W. C. (2005). Ascidian prickle regulates bothmediolateral and anterior-posterior cell polarity of notochord cells. Curr. Biol. 15, 79-85.

Kalachikov, S., Evgrafov, O., Ross, B., Winawer, M., Barker-Cummings, C.,Martinelli Boneschi, F., Choi, C., Morozov, P., Das, K., Teplitskaya, E. et al. (2002).Mutations in LGI1 cause autosomal-dominant partial epilepsy with auditory features.Nat. Genet. 30, 335-341.

Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995).Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253-310.

Lin, S., Baye, L. M., Westfall, T. A. and Slusarski, D. C. (2010). Wnt5b-Ryk pathwayprovides directional signals to regulate gastrulation movement. J. Cell Biol. 190, 263-278.

Link, B. A., Fadool, J. M., Malicki, J. and Dowling, J. E. (2000). The zebrafish youngmutation acts non-cell-autonomously to uncouple differentiation from specificationfor all retinal cells. Development 127, 2177-2188.

Macdonald, R. L. and Barker, J. L. (1977). Pentylenetetrazol and penicillin areselective antagonists of GABA-mediated post-synaptic inhibition in culturedmammalian neurones. Nature 267, 720-721.

Mapp, O. M., Wanner, S. J., Rohrschneider, M. R. and Prince, V. E. (2010). Prickle1bmediates interpretation of migratory cues during zebrafish facial branchiomotorneuron migration. Dev. Dyn. 239, 1596-1608.

Mapp, O. M., Walsh, G. S., Moens, C. B., Tada, M. and Prince, V. E. (2011). ZebrafishPrickle1b mediates facial branchiomotor neuron migration via a farnesylation-dependent nuclear activity. Development 138, 2121-2132.

Mrkusich, E. M., Flanagan, D. J. and Whitington, P. M. (2011). The core planar cellpolarity gene prickle interacts with flamingo to promote sensory axon advance inthe Drosophila embryo. Dev. Biol. 358, 224-230.

Mulley, J. C., Scheffer, I. E., Petrou, S. and Berkovic, S. F. (2003). Channelopathies asa genetic cause of epilepsy. Curr. Opin. Neurol. 16, 171-176.

Narimatsu, M., Bose, R., Pye, M., Zhang, L., Miller, B., Ching, P., Sakuma, R., Luga,V., Roncari, L., Attisano, L. et al. (2009). Regulation of planar cell polarity by Smurfubiquitin ligases. Cell 137, 295-307.

Ng, J. (2012). Wnt/PCP proteins regulate stereotyped axon branch extension inDrosophila. Development 139, 165-177.

Nishimura, D. Y., Baye, L. M., Perveen, R., Searby, C. C., Avila-Fernandez, A.,Pereiro, I., Ayuso, C., Valverde, D., Bishop, P. N., Manson, F. D. et al. (2010).Discovery and functional analysis of a retinitis pigmentosa gene, C2ORF71. Am. J.Hum. Genet. 86, 686-695.

Park, H. C., Kim, C. H., Bae, Y. K., Yeo, S. Y., Kim, S. H., Hong, S. K., Shin, J., Yoo, K.W., Hibi, M., Hirano, T. et al. (2000). Analysis of upstream elements in the HuCpromoter leads to the establishment of transgenic zebrafish with fluorescentneurons. Dev. Biol. 227, 279-293.

Patrylo, P. R., Browning, R. A. and Cranick, S. (2006). Reeler homozygous miceexhibit enhanced susceptibility to epileptiform activity. Epilepsia 47, 257-266.

Piper, R. C. and Lehner, P. J. (2011). Endosomal transport via ubiquitination. TrendsCell Biol. 21, 647-655.

Poduri, A. and Lowenstein, D. (2011). Epilepsy genetics; past, present, and future.Curr. Opin. Genet. Dev. 21, 325-332.

Pretorius, P. R., Baye, L. M., Nishimura, D. Y., Searby, C. C., Bugge, K., Yang, B.,Mullins, R. F., Stone, E. M., Sheffield, V. C. and Slusarski, D. C. (2010).Identification and functional analysis of the vision-specific BBS3 (ARL6) long isoform.PLoS Genet. 6, e1000884.

Sanchez-Alvarez, L., Visanuvimol, J., McEwan, A., Su, A., Imai, J. H., and Colavita,A. (2011). VANG-1 and PRKL-1 cooperate to negatively regulate neurite formation inCaenorhabditis elegans. PLoS Genet. 7, e1002257.

Schmitt, E. A. and Dowling, J. E. (1996). Comparison of topographical patterns ofganglion and photoreceptor cell differentiation in the retina of the zebrafish, Daniorerio. J. Comp. Neurol. 371, 222-234.

Scita, G. and Di Fiore, P. P. (2010). The endocytic matrix. Nature 463, 464-473.Shimojo, M. and Hersh, L. B. (2003). REST/NRSF-interacting LIM domain protein, a

putative nuclear translocation receptor. Mol. Cell Biol. 23, 9025-9031.Shimojo, M. and Hersh, L. B. (2006). Characterization of the REST/NRSF-interacting

LIM domain protein (RILP): localization and interaction with REST/NRSF. J.Neurochem. 96, 1130-1138.

Striano, P., Busolin, G., Santulli, L., Leonardi, E., Coppola, A., Vitiello, L., Rigon, L.,Michelucci, R., Tosatto, S. C., Striano, S. et al. (2011). Familial temporal lobeepilepsy with psychic auras associated with a novel LGI1 mutation. Neurology 76,1173-1176.

Takeuchi, M., Nakabayashi, J., Sakaguchi, T., Yamamoto, T. S., Takahashi, H.,Takeda, H. and Ueno, N. (2003). The prickle-related gene in vertebrates is essentialfor gastrulation cell movements. Curr. Biol. 13, 674-679.

Tao, H., Manak, J. R., Sowers, L., Mei, X., Kiyonari, H., Abe, T., Dahdaleh, N. S.,Yang, T., Wu, S., Chen, S. et al. (2011). Mutations in prickle orthologs cause seizuresin flies, mice, and humans. Am. J. Hum. Genet. 88, 138-149.

Teng, Y., Xie, X., Walker, S., Rempala, G., Kozlowski, D. J., Mumm, J. S. andCowell, J. K. (2010). Knockdown of zebrafish Lgi1a results in abnormal development,brain defects and a seizure-like behavioral phenotype. Hum. Mol. Genet. 19, 4409-4420.

Teng, Y., Xie, X., Walker, S., Saxena, M., Kozlowski, D. J., Mumm, J. S. and Cowell, J.K. (2011). Loss of zebrafish lgi1b leads to hydrocephalus and sensitization topentylenetetrazol induced seizure-like behavior. PLoS ONE 6, e24596.

Thisse, C., Thisse, B., Schilling, T. F. and Postlethwait, J. H. (1993). Structure of thezebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutantembryos. Development 119, 1203-1215.

Tissir, F. and Goffinet, A. M. (2010). Planar cell polarity signaling in neuraldevelopment. Curr. Opin. Neurobiol. 20, 572-577.

Tree, D. R. P., Shulman, J. M., Rousset, R., Scott, M. P., Gubb, D. and Axelrod, J. D.(2002). Prickle mediates feedback amplification to generate asymmetric planar cellpolarity signaling. Cell 109, 371-381.

Disease Models & Mechanisms 687

PCP in epilepsy and retina neurogenesis RESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

Veeman, M. T., Slusarski, D. C., Kaykas, A., Louie, S. H. and Moon, R. T. (2003).Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulatesgastrulation movements. Curr. Biol. 13, 680-685.

Velisek, L., Kubova, H., Pohl, M., Stankova, L., Mareš, P. and Schickerova, R. (1992).Pentylenetetrazol-induced seizures in rats: an ontogenetic study. NaunynSchmiedebergs Arch. Pharmacol. 346, 588-591.

Wada, H., Tanaka, H., Nakayama, S., Iwasaki, M. and Okamoto, H. (2006). Frizzled3a and Celsr2 function in the neuroepithelium to regulate migration of facialmotor neurons in the developing zebrafish hindbrain. Development 133, 4749-4759.

Wang, Y., Dye, C. A., Sohal, V., Long, J. E., Estrada, R. C., Roztocil, T., Lufkin, T.,Deisseroth, K., Baraban, S. C. and Rubenstein, J. L. (2010a). Dlx5 and Dlx6regulate the development of parvalbumin-expressing cortical interneurons. J.Neurosci. 30, 5334-5345.

Wang, Y. Y., Smith, P., Murphy, M. and Cook, M. (2010b). Global expression profilingin epileptogenesis: does it add to the confusion? Brain Pathol. 20, 1-16.

Westerfield, M. (1993). The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish(Brachydanio rerio). Eugene, OR: M. Westerfield.

Zhou, L., Bar, I., Achouri, Y., Campbell, K., De Backer, O., Hebert, J. M., Jones, K.,Kessaris, N., de Rouvroit, C. L., O’Leary, D. et al. (2008). Early forebrain wiring:genetic dissection using conditional Celsr3 mutant mice. Science 320, 946-949.

dmm.biologists.org688

PCP in epilepsy and retina neurogenesisRESEARCH ARTICLED

iseas

e M

odel

s & M

echa

nism

s

DM

M

SUMMARYINTRODUCTIONTRANSLATIONAL IMPACTRESULTSpk1a knockdown sensitizes zebrafish to PTZ treatmentpk1a knockdown induces neurite outgrowth in the retinaPk1a epilepsy mutant forms display distinct functional and biochemical properties

DISCUSSIONMATERIALS AND METHODSEthical statementZebrafish maintenanceMorpholinos, expression constructs and primersDrug treatment and motility assayIn situ hybridizationRetina histology and fluorescence microscopyWestern blot and pull-down assayVision startle response assay

Supplementary material