knockdown of zebrafish blood vessel epicardial substance results in incomplete retinal lamination

Research ArticleKnockdown of Zebrafish Blood Vessel Epicardial SubstanceResults in Incomplete Retinal Lamination

Yu-Ching Wu1 Ruei-Feng Chen1 Chia-Yang Liu2 Fung Rong Hu3

Chang-Jen Huang4 and I-Jong Wang3

1 Department of Life Science College of Life Science National Taiwan University Taipei 10617 Taiwan2Department of Ophthalmology Graduate Program of Neuroscience University of Cincinnati College of Medicine CincinnatiOH 45267 USA

3Department of Ophthalmology National Taiwan University Hospital Taipei 10002 Taiwan4 Institute of Biological Chemistry Academia Sinica Taipei 11529 Taiwan

Correspondence should be addressed to Ruei-Feng Chen rfchenntuedutw and I-Jong Wang ijongms8hinetnet

Received 18 December 2013 Accepted 29 January 2014 Published 6 March 2014

Academic Editors A M Berrocal and Y Zhong

Copyright copy 2014 Yu-Ching Wu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in theretina In vertebrates blood vessel epicardial substance (Bves) is known to play an important role in the formation andmaintenanceof the tight junctions essential for epithelial cell polarity In the current study we generated a transgenic zebrafish Bves (zbves)promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinallamination Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were usedto identify the morphological defects in normal and Bves knockdown zebrafish In normal zebrafish Bves is located at the apicaljunctions of embryonic retinal neuroepithelia during retinogenesis later it is strongly expressed around inner plexiform layer(IPL) and retinal pigment epithelium (RPE) In contrast a loss of normal retinal lamination and cellular polarity was found withundifferentiated photoreceptor cells in Bves knockdown zebrafish Herein our results indicated that disruption of Bves will resultin a loss of normal retinal lamination

1 Introduction

The vertebrate retina can be used as a model to study cellpatterning and cell fate determination within the central ner-vous system from which the retina is derived moreover theretina is easily observed and accessible during development[1] The neural retina in vertebrates differentiates between asheet of multipotent and proliferating neuroepithelial cellsundergoing a dramatic morphogenetic change that dependson a proper epithelial polarity and integrity to reshape theoriginal cell layers during retinogenesis [2] In the early stageprogenitor cells at the ventricular margin of the neural retinaundergo mitosis are divided into six classes of cells pho-toreceptors horizontal cells bipolar cells amacrine ganglioncells and Muller glia and extend their neurites leading to alaminar pattern of the retina [3]

The photoreceptor cells have both neuronal and epithelialproperties [4] Therefore cell polarity is a major featureof vertebrate photoreceptors each of which is further sub-divided into four parts in the developed retina an outersegment (OS) an inner segment (IS) a cell body (CB) anda synaptic terminus (ST) [4] The differentiation of retinalpigment epithelium (RPE) is related to the development ofphotoreceptors [5 6] Several junctional complexes includ-ing adherens junctions and tight junctions participate in thisprocess [6] Coordinating with RPE these tight connectionsin photoreceptors are able to prevent certain substances inchoroid vessels from entering the retinal tissue [6 7] Herethe establishment and regulation of junctional componentsare indispensable for the function and the integrity of RPEand photoreceptor cells in retinogenesis Most importantlythe molecular mechanism controlling cell polarity formation

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 803718 9 pageshttpdxdoiorg1011552014803718

2 The Scientific World Journal

in the retinal photoreceptor cells is interesting and importantin retinal development using an appropriate model Forexample several studies have shown that several mutationloci in zebrafish that encode proteins required for apicobasalpolarity such asmosaic eyes (moe) okomeduzy (ome) nagieoko (nok) and heart and soul (has) showed disruption ofretinal lamination [8ndash11] These studies also demonstratedthat the zebrafish is a good animal model to study the effectof junctional complexes on retinal development

The bves (blood vesselepicardial substance) gene encodesa membrane protein [12 13] and its protein has a Popeyedomainbelonging to the Popeye domainmdashcontaining (Popdc)family Bves is widely expressed in many epithelial tissuesincluding the eye [12 14ndash18] and plays a role in cell adhesionepithelial integrity and cell motility [19] During early eyedevelopment Bves is initially localized at the apical-lateralposition of epithelial precursors later on it is expressedin the retina lens and cornea [20] In vitro knockdownexperiments in a cultured corneal cell line demonstrated thatBvesmay affect epithelial cell movement during corneal reep-ithelialization [15] Our previous study further elucidated thatBves might regulate the formation of a polarized epithelialsheet through the association with the polarity protein aPKC(atypical protein kinase c) [21] Combined with its expressionpattern in the eye and its role in cell junctions we believe thatthe expressions of Bves in epithelial adhesion and movementare crucial for eye development Although we speculate thatBves should play a physiological role during eye developmentlittle is known about the role of Bves in retinal laminationand photoreceptor differentiation In this study we generateda transgenic fish line in which zbves promoter driven EGFPcould be visualized to localize its expression pattern duringeye development We further used a knockdown techniqueto study the expression of Bves during retinal lamination andphotoreceptor differentiation

2 Materials and Methods

21 Transgenic Zebrafish Line The Tg(zbves EGFP) trans-genic zebrafish line was established using the generatedconstruct T2K-zbves-p (Figure 1(a)) containing two trans-poson sequences and the 6-kb DNA of the zebrafish zbvespromotermdashconjugated EGFP sequence This transposon-donor plasmid and transposase mRNAs were coinjected intofertilized eggs and the transgenic fish line was created F1embryos exhibiting EGFP expression at normal temperatures(sim28∘C) were raised and F3 embryos were used for observa-tion in this study

22 Morpholino Knockdown The following antisense mor-pholino oligonucleotides (MOs) were purchased from GeneTools (Philomath OR) a zbves translation blocking mor-pholino (ATGmo 51015840-GATGTTGTGTTGGACATTCTGA-GGC-31015840) and a zbves splice inhibition morpholino(splice mo 51015840-AGAGCAGCCTGAAAGACAATAAAGA-31015840) Zebrafish embryos at the 1- or 2-cell stages were chosenfor injection We chose 2 ng of ATGmo and 4 ng of splice moin subsequent experiments These concentrations resulted

in 90 survival from injected embryos with more than 50exhibiting a phenotype [21]

23 Immunofluorescence Staining For immunofluorescencedetection of proteins on cryosections of embryos stagedembryos were fixed in 4paraformaldehyde at 4∘Covernightand then transferred to 30 sucrose After overnight immer-sion in sucrose embryos were embedded within OCT(Tissue-Tek) and stored at ndash80∘C 20120583m cryosections werecut and loaded on glass slides Then these samples werewashed in Triton X-100 for 30min at room temperature andthen blocked in 5 BSA at 4∘C overnight The embryoswere incubated with the following primary antibodies inblocking buffer at 4∘C overnight affinity purified anti-zBves(1 500) [21] anti-ZO-1 (1 200 Zymed S San Francisco CA)anti-PKC120577 (1 200 Santa Cruz Biotechnology Santa CruzCA) zn8 (1 50 Zebrafish International Resource CenterEugene OR) anticarbonic anhydrase II (CA 1 100 AbcamCambridge England) antityrosine hydroxylase (TH 1 100Millipore Temecula CA) anti-CtBP (1 200 Santa CruzBiotechnology Santa Cruz CA) anti-Crx (1 200 Santa CruzBiotechnology Santa Cruz CA) and zpr-1 (1 400 ZebrafishInternational Resource Center Eugene OR) Embryos werewashed in PT1 BSA and then appropriate secondaryantibodies (ie Alexa 488 1 200 Invitrogen Carlsbad CA)were added for 2 h at room temperature Samples werewashed and images were acquired using confocal microscopy(LSM510 Meta Zeiss Thornwood NY)

24 ElectronMicroscopy At 96 hpf the controls and the zbvesATGmo-injected embryos were immersed in a fixative solu-tion containing 15 glutaraldehyde and 15 paraformalde-hyde in 01M cacodylate buffer (pH 74) for 2 h at roomtemperature After a wash in 01M cacodylate buffer thesamples were postfixed in 02 osmium tetroxide in 01Mcacodylate buffer for 16ndash18 h The samples were then rinsedin the buffer dehydrated in a graded series of ethanol andembedded in Epon 812 1m sections were then observed aftertoluidine blue staining Thin sections were contrasted withuranyl acetate and lead citrate Grids were observed by thetransmission electron microscopy (TEM Hitachi H-7500Tokyo Japan)

3 Results

31 Spatiotemporal Expression of zBves in Zebrafish Eye duringDevelopment To examine the Bves expression pattern atransgenic zebrafish linemdashTg(zbves EGFP)mdashwas generatedusing a transposon system in which Tol2 vector was usedas previously described (Figure 1(a)) [22] The fluorescentsignal of F3 transgenic zebrafish embryos appears at 4 hpfand is highly expressed in the cells of yolk syncytial layer(YSL) (Figure 1(b)) Then the fluorescence spreads to theenvelope layer (EVL) at 8 hpf (Figure 1(c)) Before formingthe optic vesicle at 12 hpf the eye field begins to evaginateand the fluorescence significantly appears in the epider-mis layer (Figure 1(d)) At 18 hpf when the optic cup isformed the fluorescent signals concentrate at the anterior

The Scientific World Journal 3

Tol2 Tol2zBves promoter YSL

EVLEye In

ONL

(a) (b)

(e)(d)(c)

(g)

(h) (i)

(f)

In

InIn In

GCLONL

ONL

INL

IPL

nr

nr

ONL

INL

IPL

EGFP

(e998400)

(g998400)

(f 998400)

(g998400998400)

8hpf

4hpf

24 hpf

24 hpf 24 hpf 96 hpf

96 hpf96 hpf

12 hpf 18 hpf

(i998400)

Figure 1 ZBves expression in the eyes of transgenic lineTheTol2 transposon systemwas established to produce a Tg(zbves EGFP) transgeniczebrafish line The zbves promoter sequences were cloned in the Tol2-containing vector (a) The F3 transgenic embryos were observed usinga confocal microscope to investigate the expression pattern of Bves Before 18 hpf the GFP signal (transgene) mainly appeared on the surfaceof whole organism and eye (b)ndash(d) At 18 hpf Bves expression initially spread into the retina from the border between the cornea and retina(e) (e1015840) At 24 hpf zBves was expressed in whole retina of the zebrafish eye (f) (f rsquo) At 96 hpf zBves was localized around IPL and RPE (g)(g1015840) and (g10158401015840) Antibody staining with Bves was preformed in 24-hpf 96-hpf and adult fish (h) (i) and (i1015840) The lateral side of the eye withphalloidin staining (red) is shown in (e1015840) (f1015840) (g1015840) and (g10158401015840) Scale bar 50 120583m

margin of retina in the eye field (Figures 1(e) and 1(e1015840))At 24 hpf it can be spread to whole eye tissue (Figures 1(f)and 1(f1015840)) At 96 hpf Bves expressed cells were localizedaround inner plexiform layer (IPL) and RPE (Figures 1(g)1(g1015840) and 1(g10158401015840))

We used an anti-zBves antibody to confirm the distri-bution and location of the Bves protein in 24 hpf 96 hpf

and adult fish Compared with the fluorescent signals in thetransgenic embryos the expression pattern of Bves proteinis compatible with the fluorescent pattern in transgeniczebrafish (Figures 1(f1015840) 1(g1015840) 1(g10158401015840) 1(h) 1(i) and 1(i1015840)) At24 hpf the Bves protein is localized at the apical regionof the retinal epithelium (Figure 2(i)) Compared with theadherens junction-associated actin bundles colocalized with


In In

(a) (b) (c)

(d)

(e)

(g) (h)

(i) (j) (k) (l)

(f)

WT

IPLONL

OPL

IPL

04

03

02

01

001

(dpf)

WT-whole eyeATGmo-whole eye

WT-lensATGmo-lens

2 3

lowast

lowast

lowast

lowastlowast

lowast

Leng

th (m

m)

3dpf ATGmo 3dpf Splice mo 3dpf

WT 3dpf

WT 4dpf

WT 1dpf

ATGmo 3dpf

ATGmo 4dpf

ATGmo 1dpf WT 4dpf ATGmo 4dpf

Figure 2 Zbves morphants exhibited defects in eye size and lamination The morphological observations of wild-type and ATGmo- andsplice mo-injected embryos are shown Compared with wild-type (a) the eyes formed abnormally and resulted in microphthalmos afterknockdown of zBves (b) (c) Statistical analyses of the length of whole eye and lens in both groups were performed (119899 = 11 t-test for twogroups 119875 lt 001) (d) HE staining in paraffin sections of wild-type and morphant embryos revealed the eye without a laminated retina (e)(f) The same patterns were observed in the semithin Epon sections with toluidine blue staining (g) (h) Scale bar 50120583m Immunostainingwith anti-zBves antibody (green) and phalloidin (red) was performed in the retinal neuroepithelia of wild-type (i) and zBves MO-treatedembryos (j) at 24 hpf and laminated retinas of untreated (k) and zBves MO-treated embryos (l) at 96 hpf ln lens Scale bar 10120583m

phalloidin high level Bves expression is at apical sites of retinaepithelium (Figure 2(i))

32 Bves Knockdown Results in Microphthalmos with RetinalLamination Defect To examine the physiological functionof Bves in eye development two kinds of morpholinooligonucleotides (MOs)mdashATGmo and splicing momdashwereused to disrupt the production of the zBves protein In

our previous study zBves knockdown embryos exhibitedepidermal defects In addition a dramatically small eyefield could be observed in morphants (Figure 2) Comparedwith the untreated embryos MO-injected embryos havesignificantly small eyes (Figures 2(a)ndash2(c)) The thicknessesof lenses and the lengths of whole eyes were measured andtheir differences were found to be statistically different at 1 2and 3 dpf respectively (Figure 2(d))


Wild

type

Zn8

Zn8

CALL

CALL

TH

TH

Zpr-1

Zpr-1

Ganglion neurons

(a)

(e) (f) (g) (h)

(b) (c) (d)

(i) (j)

(k) (l)

(m) (n)

Muller gliaInterplexiform

neuronsDouble cone

photoreceptors

lowast

lowast lowastlowast

lowastlowast

50120583m

WT

WT

WT

CtBP

zpr

-1Cr

x

1000nmATGmo (333)

ATGmo (375)

ATG

mo

ATGmo

NN

(m998400) (n998400)

Figure 3 Disruption of zBves affected the dysregulated patterns in the retina The frozen sections and immunofluorescence staining withdifferent marker antibodiesmdashzn8 for ganglion cells (a) (e) anti-CAII for Muller glia (b) (f) anti-TH for interplexiform neurons (c) (g) andzpr-1 for photoreceptors (d) (h) were performed in eyes of 96 hpf wild-type and zBves MO-injected embryos Immunofluorescence stainingwith Crx (i) (j) antibody in the frozen sections was performed in 72 hpf embryos 375 (616) of mutant retina showed Crx protein Theultrastructures of the retina in wild-type (m) (m1015840) and zBves MO-injected embryos (n) (n1015840) were analyzed by TEM Double staining withCtBP and zpr-1 was adopted in frozen crytosections (k) (l) 333 (412) of mutant retina showed CtBP protein without zpr-1 expression(l) Asterisks indicate the outer segment Black arrows indicate the inner segment N nucleus S Scale bar in immunostaining photographs50 120583m Scale bar in TEM picture 1000 nm

To further compare the microstructural differencesbetween untreated and zBves knockdown embryos wefound that the morphants had a microphthalmos withouta laminated retina through hematoxylin and toluidine bluestaining (Figures 2(e)ndash2(h)) Additionally there was a spacebetween the retina and the RPE indicating subretinal fluidretention (Figures 2(g) and 2(h)) This indicates that zBvesdisruption might break the RPE barrier in retina and impliesthat Bves might play an important role in the precise for-mation of retinal layer structure during retinogenesis Theexpressions of actin and Bves were examined in morphants

and revealed an unclosed apical pattern of phalloidin-stainedadherens junction-associated actin bundles (Figure 2(j))This result implies that Bves is involved in the generation ormaintenance of epithelial cell polarity like that in the epitheliaof other tissues [21 23] Furthermore we examined theexpression of Bves after retinal lamination Interestingly at96 hpf Bves was found in the cell membranes of all epitheliallayers of the eye however the retina exhibited the strongestexpression In the retina Bveswas expressed near RPE barrierand around the inner plexiform layer (Figure 2(k)) whichis composed of the amacrine cells and the ganglion cells


OLM

OPL

ZO-1

WT

(a)

ZO-1

ATGmo

(b)

ZO-1

Splice mo

(c)

ZO-1

ATGmo + bves

(d)

aPKC

ONL

(e)

aPKC

(f)

aPKC

(g)

aPKC

(h)

ONL

120573-Catenin

(i)

120573-Catenin

(j)

120573-Catenin

(k)

10120583m 120573-Catenin

(l)

Figure 4 Adherens junctions were not formed in the outer limiting membrane of zbves morphants Immunostaining of adherensjunction-related molecules-ZO-1 (a)ndash(d) aPKC (e)ndash(h) and 120573-catenin (i)ndash(l)mdashwas carried out in the retinas of wild-type and zBves MO-injected embryos and zbvesmRNA rescued morphants at 72 hpf In wild-type embryos 120573-catenin obviously appeared in the apical site ofphotoreceptors (i) whereas it disappeared in zbve morphants (j) (k) The defect in this protein among morphants could be rescued bycoinjection with zbvemRNA Arrows indicate the apical edge Nuclei were counterstained with DAPI (blue) Scale bar 10 120583m

Furthermore the bundles of actin in zBves knockdown retinawere dramatically disrupted (Figure 2(l)) These suggest thatBves might play a role in cell-cell junctional maintenanceespecially at the apical side of the RPE membrane after eyeformation as described previously [21]

33 Incomplete Photoreceptor Formation in Bves MorphantsBecause has encodes atypical protein kinase C whichrequires Bves to regulate cell junction and the zebrafish hasmutant exhibits loss of normal retinal lamination [24] wefurther investigated whether Bves is required for retinal lam-inationThe retinae of morphants were stained with differentretinal cell-specific antibodies to identify the differentiationof retinal cells in lamination defect during eye developmentOur results showed that ganglion cells Muller glia andinterplexiform cells were present in both morphants (Figures3(e)ndash3(g)) however most of them were mislocalized and

unlike those of the normal retina (Figures 3(a)ndash3(c)) Inter-estingly immunostaining with the zpr-1 antibody revealedthat photoreceptors are not present at 96 hpf after Bvesknockdown (asterisks in Figure 5(h)) whereas they exist inwild-type embryos (arrows in Figure 3(d)) To confirm theseresults cone-rod homebox-containing gene (Crx) in relationwith eye development was further observed in 72-hpf retina(Figure 3(i)) Surprisingly Crx protein did not disappear inall mutant retinae 375 of mutant retina expressed Crxprotein (Figure 3(j)) but 625 of them did not These resultsindeed demonstrate that Bves is required for proper retinallamination especially for photoreceptors

To examine themorphology of photoreceptor and to con-firm the result of zpr-1 and Crx staining the ultrastructuresand CtBP (synaptic ribbon) expression of the photoreceptorswere analyzed by TEM and immunostaining Compared withthe microstructure of a normal retina (Figures 3(m) and3(m1015840)) zbves morphants were found to lack outer segments


RGC

IPL

INL

OPL

ONL

RPE

Bves knockdown

A loss of normal

retinal lamination

Photoreceptor

defect

Figure 5 Illustration of Bves effect on zebrafish retinogenesis RGC retinal ganglion cells IPL inner plexiform layer INL inner nuclearlayer OPL outer plexiform layer ONL outer nuclear layer RPE retinal pigment epithelium Red spot indicates tight junction

under transmission electronic microscopy (Figures 3(n)and 3(n1015840)) 333 of mutant retina had CtBP expression(Figure 3(l)) and 666 of them did not show CtBP pro-tein whereas all wild-type retinae showed CtBP protein(Figure 3(k)) These results indicate that zBves knockdownresults in immature and even undifferentiated photorecep-tors

34 Bves is Essential for the Formation of Cell Polarity betweenRetina Neuron and RPE to Assist in the Differentiation ofPhotoreceptor Cells As Bves is localized in adherens junc-tions we examined whether the expression patterns of ZO-1 (zona occludens protein 1) aPKC and 120573-catenin whichare initially associated with adherens junctions in the RPEare altered after Bves knockdown The strongest expressionsof ZO-1 aPKC and 120573-catenin are normally observed at theapical surface of the photoreceptor cells and the RPE in auniformly honeycomb-like pattern (Figures 4(a) 4(e) and4(i)) In Bves knockdown embryos however the patterns ofZO-1 aPKC and 120573-catenin were found to be discontinuousalong the apical surface of photoreceptor cells and formedinto rosette structures (Figures 4(b) 4(c) 4(f) 4(g) 4(j) and4(k)) This defect could be rescued by coinjection with zbvesmRNA in morphants (Figures 4(d) 4(h) and 4(l)) Theseresults indicate that Bves is crucial for the proper localizationof adherens junction proteins along the apical border of thephotoreceptor cells and RPE

4 Discussion

During eye development the formation of different ocularlayers relies on the tight junctions to anchor the changingtissue shape and cellular polarities [25] and the function ofBves in tight junctions has also been confirmed in this process

and described in many studies [21 23 26] In our studywe found that Bves can be expressed earlier in single-layerepithelia of the EVL and later expressed in the ocular surfaceepithelium lens and retina of the eye (Figure 1) At 24 hpfBves spreads throughout the eye tissue and locates at cellplasmamembranes (Figure 2(i)) At that time neuroepitheliain eyes are already migrating to the eye cup these thenproliferate or differentiate according to the signals from theirniches [27] Therefore according to the expression patternwe consider that Bves might play a role in assisting andregulating the epithelial cell shape and polarity during earlyeye development

Interestingly Bves is strongly expressed in the apicalsite of neuroepithelia and the retina (Figure 2(k)) whichcontact with RPE before closure of the subretinal space andform an outer limiting membrane and a retina-blood barrier[28] Our recent in vivo studies have shown that Bves canregulate paracellular permeability in epithelia [21] Thus itis reasonable to speculate that Bves may be involved in theregulation of the blood-retina barrier and the formationof the outer limiting membrane At 4 dpf zbves morphantsshow fluid accumulation between the RPE and the retina(Figure 2(h)) We further reason that this fluid accumulationresults from the disruption of the blood-retina barrier

After Bves knockdown the eye becomes smaller and losesits normal arrangement as in microphthalmia (Figures 2(b)and 2(c)) Many transcription and regulatory factors suchas Pax6 Crx Otx2 MITF Sox2 and shh are involved ineye development and the mutation of these factors will alsoinduce microphthalmia [29] Although the cause of small eyein these mutants is still unclear [29] these studies showedthe importance of the lens in regulating eye growth inwhich ablation of the lens or mutation of genes vital forlens development leads to microphthalmia [30] In our studywe also showed poorly arranged lenticular fibers in the lens


at 3 dpf (Figure 2(i)) Therefore it is possible to speculatethat the microphthalmia could result from the disruptionof lenticular formation in zbves morphants and it will be ofinterest to investigate the possible causes of microphthalmiain future study In addition we find that Crx protein expres-sions in zBves mutants are disordered and even disappeared(Figures 3(i) and 3(j))These imply that defects in laminationand photoreceptor development in zBves knockdown retinaemight result from abnormal transcriptions factor expressionsin early stage which arises from the zBves knockdown anddisruption of cell polarity

The outer blood-retinal barrier lies between RPE and theouter surface of the photoreceptor layer [6] To form thisbarrier a belt of cell junctions around the apical domain of thephotoreceptor should be formed to support the cell polarityof photoreceptors and to form a barrier between the choroidand the retina [6] Studies on Drosophila have indicated thatgenetic regulators of photoreceptor polarity are similar to thegenes regulating cell polarity in other epithelial tissue [31ndash33]The analysis of the vertebrate eye also lends credenceto the idea that the regulators of photoreceptor polar-ity are related to epithelial polarity pathways [34 35]Our results confirm this notion and indicate that Bves isalso crucial for the proper localization of adherens junc-tion proteins along the apical border of the photoreceptorcells and RPE

The most important thing in our study is that zbvesmorphants lost their retinal lamination In neural tissuea proper differentiation of apical and basal domains inneuroepithelial cells determines cell fate and laminationduring development [1] Because Bves is located in theapical regions in the retina and plays a role in cellpolarity we think that Bves knockdown would disruptnormal cell polarity during retinogenesis Furthermoreimmunostaining with specific retinal cell makers showedthat retinal cell differentiation in zbves morphants was notaffected except in the photoreceptor cells (Figure 3(h))most of which revealed an undifferentiated pattern withouta normal outer segment formation (Figures 3(n) and 3(n1015840))Koike et al indicated that polarized photoreceptors couldassist progenitors to anchor at the apical edge of theretina which led to the formation of a correct laminarretina [36] Therefore we suggest that Bves is involvedin retinal lamination through polarized photoreceptorsFurthermore the bundles of junction proteins betweenretina and RPE in Bves knockdown embryos revealed adisordered and a discontinuous arrangement (Figures 4(b)4(c) 4(f) 4(g) 4(j) and 4(k)) and zbves mRNA couldrescue these defects (Figures 4(d) 4(h) and 4(l))Taking them all together our results suggest that Bvescan regulate photoreceptor differentiation in retinallamination (Figure 5)

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

We would like to thank Ya-Wen Hsu for her help throughoutthis study and the second core lab of National TaiwanUniversity Hospital for the supply of space and instrumentsWe would also like to thank Ms Hui-Chun Kung and Ya-Ling Chen for their help throughout the TEM image study atthe Microscope Center of Chang-Gung Memorial HospitalWe appreciated the help from Ji-Ying Huang at NationalTaiwan University Hospital Image Core Lab for the technicalassistance in confocol images The studies were supported inpart by NSC Grantsmdashnos 983112B002040 993112B002029and 992314B002039MY3mdashand Grant from NTUH no 98S-1105

References

[1] A London I Benhar and M Schwartz ldquoThe retina as awindow to the brain-from eye research to CNS disordersrdquoNature Reviews Neurology vol 9 pp 44ndash53 2013

[2] O Randlett C Norden and W A Harris ldquoThe vertebrateretina a model for neuronal polarization in vivordquo Developmen-tal Neurobiology vol 71 no 6 pp 567ndash583 2011

[3] E Willbold ldquoMuller glia cells and their possible roles duringretina differentiation in vivo and in vitrordquo Histology andHistopathology vol 13 no 2 pp 531ndash552 1998

[4] J N Pearring R Y Salinas S A Baker and V Y ArshavskyldquoProtein sorting targeting and trafficking in photoreceptorcellsrdquo Progress in Retinal and Eye Research vol 36 pp 24ndash512013

[5] O L German E Buzzi N P Rotstein E Rodrıguez-Boulanand L E Politi ldquoRetinal pigment epithelial cells promote spatialreorganization and differentiation of retina photoreceptorsrdquoJournal of Neuroscience Research vol 86 no 16 pp 3503ndash35142008

[6] L J Rizzolo S Peng Y Luo and W Xiao ldquoIntegration of tightjunctions and claudins with the barrier functions of the retinalpigment epitheliumrdquo Progress in Retinal and Eye Research vol30 no 5 pp 296ndash323 2011

[7] K Hosoya andM Tachikawa ldquoThe inner blood-retinal barriermolecular structure and transport biologyrdquo Advances in Exper-imental Medicine and Biology vol 763 pp 85ndash104 2012

[8] S Horne-Badovinac D Lin S Waldron et al ldquoPositionalcloning of heart and soul reveals multiple roles for PKC120582 inzebrafish organogenesisrdquo Current Biology vol 11 no 19 pp1492ndash1502 2001

[9] A M Jensen and M Westerfield ldquoZebrafish mosaic eyes is anovel FERM protein required for retinal lamination and retinalpigmented epithelial tight junction formationrdquoCurrent Biologyvol 14 no 8 pp 711ndash717 2004

[10] J Malicki and W Driever ldquooko meduzy mutations affectneuronal patterning in the zebrafish retina and reveal cell-cellinteractions of the retinal neuroepithelial sheetrdquo Developmentvol 126 no 6 pp 1235ndash1246 1999

[11] X Wei and J Malicki ldquoNagie oko encoding a MAGUK-familyprotein is essential for cellular patterning of the retinardquo NatureGenetics vol 31 no 4 p 439 2002

[12] B Andree T Hillemann G Kessler-Icekson et al ldquoIsolationand characterization of the novel Popeye gene family expressedin skeletal muscle and heartrdquo Developmental Biology vol 223no 2 pp 371ndash382 2000


[13] D E Reese M Zavaljevski N L Streiff and D Baderldquobves a novel gene expressed during coronary blood vesseldevelopmentrdquo Developmental Biology vol 209 no 1 pp 159ndash171 1999

[14] M E Osler and D M Sader ldquoBves expression during avianembryogenesisrdquo Developmental Dynamics vol 229 no 3 pp658ndash667 2004

[15] A N Ripley M S Chang and D M Bader ldquoBves is expressedin the epithelial components of the retina lens and corneardquoInvestigative Ophthalmology and Visual Science vol 45 no 8pp 2475ndash2483 2004

[16] T K Vasavada J R DiAngelo and M K Duncan ldquoDevelop-mental expression of Pop1Bvesrdquo Journal of Histochemistry andCytochemistry vol 52 no 3 pp 371ndash377 2004

[17] T K Smith and D M Bader ldquoCharacterization of Bvesexpression during mouse development using newly generatedimmunoreagentsrdquoDevelopmental Dynamics vol 235 no 6 pp1701ndash1708 2006

[18] A Torlopp S S Breher J Schluter and T Brand ldquoComparativeanalysis of mRNA and protein expression of Popdc1 (Bves)during early development in the chick embryordquo DevelopmentalDynamics vol 235 no 3 pp 691ndash700 2006

[19] M A Amato S Boy and M Perron ldquoHedgehog signaling invertebrate eye development a growing puzzlerdquo Cellular andMolecular Life Sciences vol 61 no 7-8 pp 899ndash910 2004


[21] Y C Wu C Y Liu Y H Chen R F Chen C J Huang andI J Wang ldquoBlood vessel epicardial substance (Bves) regulatesepidermal tight junction integrity through atypical proteinkinase Crdquo The Journal of Biological Chemistry vol 287 pp39887ndash39897 2012

[22] M L Suster H Kikuta A Urasaki K Asakawa and KKawakami ldquoTransgenesis in Zebrafish with theTol2 transposonsystemrdquoMethods in Molecular Biology vol 561 pp 41ndash63 2009

[23] M E Osler M S Chang and D M Bader ldquoBves modulatesepithelial integrity through an interaction at the tight junctionrdquoJournal of Cell Science vol 118 no 20 pp 4667ndash4678 2005

[24] S Cui C Otten S Rohr S Abdelilah-Seyfried and B A LinkldquoAnalysis of aPKC120582 and aPKC120577 reveals multiple and redundantfunctions during vertebrate retinogenesisrdquo Molecular and Cel-lular Neuroscience vol 34 no 3 pp 431ndash444 2007

[25] L J Rizzolo ldquoDevelopment and role of tight junctions in theretinal pigment epitheliumrdquo International Review of Cytologyvol 258 pp 195ndash234 2007

[26] P K Russ C J Pino C SWilliams DM Bader F R Haseltonand M S Chang ldquoBves modulates tight junction associatedsignalingrdquo PLoS ONE vol 6 no 1 Article ID e14563 2011

[27] S Fuhrmann ldquoEye morphogenesis and patterning of the opticvesiclerdquoCurrent Topics in Developmental Biology vol 93 pp 61ndash84 2010

[28] D S Williams K Arikawa and T Paallysaho ldquoCytoskeletalcomponents of the adherens junctions between the photore-ceptors and the supportiveMuller cellsrdquo Journal of ComparativeNeurology vol 295 no 1 pp 155ndash164 1990

[29] T M Bardakjian and A Schneider ldquoThe genetics of anoph-thalmia and microphthalmiardquo Current Opinion in Ophthalmol-ogy vol 22 no 5 pp 309ndash313 2011

[30] A J Coulombre and J L Coulombre ldquoLens development IRole of the lens in eye growthrdquo The Journal of experimentalzoology vol 156 pp 39ndash47 1964

[31] M Mishra M Rentsch and E Knust ldquoCrumbs regulatespolarity and prevents light-induced degeneration of the simpleeyes of Drosophila the ocellirdquo European Journal of Cell Biologyvol 91 pp 706ndash716 2012

[32] S J Warner and G D Longmore ldquoDistinct functions forRho1 in maintaining adherens junctions and apical tension inremodeling epitheliardquo Journal of Cell Biology vol 185 no 6 pp1111ndash1125 2009

[33] A Djiane S Yogev and M Mlodzik ldquoThe apical determinantsaPKC and dPatj regulate frizzled-dependent planar cell polarityin the Drosophila eyerdquo Cell vol 121 no 4 pp 621ndash631 2005

[34] Y Omori and J Malicki ldquooko meduzy and related crumbsgenes are determinants of apical cell features in the vertebrateembryordquo Current Biology vol 16 no 10 pp 945ndash957 2006

[35] Y-C Hsu and A M Jensen ldquoMultiple domains in the CrumbsHomolog 2a (Crb2a) protein are required for regulating rodphotoreceptor sizerdquo BMC Cell Biology vol 11 article 60 2010

[36] C Koike A Nishida K Akimoto et al ldquoFunction of atypicalprotein kinase C 120582 in differentiating photoreceptors is requiredfor proper lamination of mouse retinardquo Journal of Neurosciencevol 25 no 44 pp 10290ndash10298 2005


in the retinal photoreceptor cells is interesting and importantin retinal development using an appropriate model Forexample several studies have shown that several mutationloci in zebrafish that encode proteins required for apicobasalpolarity such asmosaic eyes (moe) okomeduzy (ome) nagieoko (nok) and heart and soul (has) showed disruption ofretinal lamination [8ndash11] These studies also demonstratedthat the zebrafish is a good animal model to study the effectof junctional complexes on retinal development

The bves (blood vesselepicardial substance) gene encodesa membrane protein [12 13] and its protein has a Popeyedomainbelonging to the Popeye domainmdashcontaining (Popdc)family Bves is widely expressed in many epithelial tissuesincluding the eye [12 14ndash18] and plays a role in cell adhesionepithelial integrity and cell motility [19] During early eyedevelopment Bves is initially localized at the apical-lateralposition of epithelial precursors later on it is expressedin the retina lens and cornea [20] In vitro knockdownexperiments in a cultured corneal cell line demonstrated thatBvesmay affect epithelial cell movement during corneal reep-ithelialization [15] Our previous study further elucidated thatBves might regulate the formation of a polarized epithelialsheet through the association with the polarity protein aPKC(atypical protein kinase c) [21] Combined with its expressionpattern in the eye and its role in cell junctions we believe thatthe expressions of Bves in epithelial adhesion and movementare crucial for eye development Although we speculate thatBves should play a physiological role during eye developmentlittle is known about the role of Bves in retinal laminationand photoreceptor differentiation In this study we generateda transgenic fish line in which zbves promoter driven EGFPcould be visualized to localize its expression pattern duringeye development We further used a knockdown techniqueto study the expression of Bves during retinal lamination andphotoreceptor differentiation

2 Materials and Methods

21 Transgenic Zebrafish Line The Tg(zbves EGFP) trans-genic zebrafish line was established using the generatedconstruct T2K-zbves-p (Figure 1(a)) containing two trans-poson sequences and the 6-kb DNA of the zebrafish zbvespromotermdashconjugated EGFP sequence This transposon-donor plasmid and transposase mRNAs were coinjected intofertilized eggs and the transgenic fish line was created F1embryos exhibiting EGFP expression at normal temperatures(sim28∘C) were raised and F3 embryos were used for observa-tion in this study

22 Morpholino Knockdown The following antisense mor-pholino oligonucleotides (MOs) were purchased from GeneTools (Philomath OR) a zbves translation blocking mor-pholino (ATGmo 51015840-GATGTTGTGTTGGACATTCTGA-GGC-31015840) and a zbves splice inhibition morpholino(splice mo 51015840-AGAGCAGCCTGAAAGACAATAAAGA-31015840) Zebrafish embryos at the 1- or 2-cell stages were chosenfor injection We chose 2 ng of ATGmo and 4 ng of splice moin subsequent experiments These concentrations resulted

in 90 survival from injected embryos with more than 50exhibiting a phenotype [21]

23 Immunofluorescence Staining For immunofluorescencedetection of proteins on cryosections of embryos stagedembryos were fixed in 4paraformaldehyde at 4∘Covernightand then transferred to 30 sucrose After overnight immer-sion in sucrose embryos were embedded within OCT(Tissue-Tek) and stored at ndash80∘C 20120583m cryosections werecut and loaded on glass slides Then these samples werewashed in Triton X-100 for 30min at room temperature andthen blocked in 5 BSA at 4∘C overnight The embryoswere incubated with the following primary antibodies inblocking buffer at 4∘C overnight affinity purified anti-zBves(1 500) [21] anti-ZO-1 (1 200 Zymed S San Francisco CA)anti-PKC120577 (1 200 Santa Cruz Biotechnology Santa CruzCA) zn8 (1 50 Zebrafish International Resource CenterEugene OR) anticarbonic anhydrase II (CA 1 100 AbcamCambridge England) antityrosine hydroxylase (TH 1 100Millipore Temecula CA) anti-CtBP (1 200 Santa CruzBiotechnology Santa Cruz CA) anti-Crx (1 200 Santa CruzBiotechnology Santa Cruz CA) and zpr-1 (1 400 ZebrafishInternational Resource Center Eugene OR) Embryos werewashed in PT1 BSA and then appropriate secondaryantibodies (ie Alexa 488 1 200 Invitrogen Carlsbad CA)were added for 2 h at room temperature Samples werewashed and images were acquired using confocal microscopy(LSM510 Meta Zeiss Thornwood NY)

24 ElectronMicroscopy At 96 hpf the controls and the zbvesATGmo-injected embryos were immersed in a fixative solu-tion containing 15 glutaraldehyde and 15 paraformalde-hyde in 01M cacodylate buffer (pH 74) for 2 h at roomtemperature After a wash in 01M cacodylate buffer thesamples were postfixed in 02 osmium tetroxide in 01Mcacodylate buffer for 16ndash18 h The samples were then rinsedin the buffer dehydrated in a graded series of ethanol andembedded in Epon 812 1m sections were then observed aftertoluidine blue staining Thin sections were contrasted withuranyl acetate and lead citrate Grids were observed by thetransmission electron microscopy (TEM Hitachi H-7500Tokyo Japan)

3 Results

31 Spatiotemporal Expression of zBves in Zebrafish Eye duringDevelopment To examine the Bves expression pattern atransgenic zebrafish linemdashTg(zbves EGFP)mdashwas generatedusing a transposon system in which Tol2 vector was usedas previously described (Figure 1(a)) [22] The fluorescentsignal of F3 transgenic zebrafish embryos appears at 4 hpfand is highly expressed in the cells of yolk syncytial layer(YSL) (Figure 1(b)) Then the fluorescence spreads to theenvelope layer (EVL) at 8 hpf (Figure 1(c)) Before formingthe optic vesicle at 12 hpf the eye field begins to evaginateand the fluorescence significantly appears in the epider-mis layer (Figure 1(d)) At 18 hpf when the optic cup isformed the fluorescent signals concentrate at the anterior



EVLEye In

ONL

(a) (b)

(e)(d)(c)

(g)

(h) (i)

(f)

In

InIn In

GCLONL

ONL

INL

IPL

nr

nr

ONL

INL

IPL

EGFP

(e998400)

(g998400)

(f 998400)

(g998400998400)

8hpf

4hpf

24 hpf


96 hpf96 hpf

12 hpf 18 hpf

(i998400)






In In

(a) (b) (c)

(d)

(e)

(g) (h)

(i) (j) (k) (l)

(f)

WT

IPLONL

OPL

IPL

04

03

02

01

001

(dpf)


WT-lensATGmo-lens

2 3

lowast

lowast

lowast

lowastlowast

lowast

Leng

th (m

m)


WT 3dpf

WT 4dpf

WT 1dpf

ATGmo 3dpf

ATGmo 4dpf







Wild

type

Zn8

Zn8

CALL

CALL

TH

TH

Zpr-1

Zpr-1

Ganglion neurons

(a)

(e) (f) (g) (h)

(b) (c) (d)

(i) (j)

(k) (l)

(m) (n)


neuronsDouble cone

photoreceptors

lowast

lowast lowastlowast

lowastlowast

50120583m

WT

WT

WT

CtBP

zpr

-1Cr

x

1000nmATGmo (333)

ATGmo (375)

ATG

mo

ATGmo

NN

(m998400) (n998400)





OLM

OPL

ZO-1

WT

(a)

ZO-1

ATGmo

(b)

ZO-1

Splice mo

(c)

ZO-1

ATGmo + bves

(d)

aPKC

ONL

(e)

aPKC

(f)

aPKC

(g)

aPKC

(h)

ONL

120573-Catenin

(i)

120573-Catenin

(j)

120573-Catenin

(k)

10120583m 120573-Catenin

(l)







RGC

IPL

INL

OPL

ONL

RPE

Bves knockdown

A loss of normal

retinal lamination

Photoreceptor

defect




4 Discussion











Acknowledgments


References








































EVLEye In

ONL

(a) (b)

(e)(d)(c)

(g)

(h) (i)

(f)

In

InIn In

GCLONL

ONL

INL

IPL

nr

nr

ONL

INL

IPL

EGFP

(e998400)

(g998400)

(f 998400)

(g998400998400)

8hpf

4hpf

24 hpf


96 hpf96 hpf

12 hpf 18 hpf

(i998400)






In In

(a) (b) (c)

(d)

(e)

(g) (h)

(i) (j) (k) (l)

(f)

WT

IPLONL

OPL

IPL

04

03

02

01

001

(dpf)


WT-lensATGmo-lens

2 3

lowast

lowast

lowast

lowastlowast

lowast

Leng

th (m

m)


WT 3dpf

WT 4dpf

WT 1dpf

ATGmo 3dpf

ATGmo 4dpf







Wild

type

Zn8

Zn8

CALL

CALL

TH

TH

Zpr-1

Zpr-1

Ganglion neurons

(a)

(e) (f) (g) (h)

(b) (c) (d)

(i) (j)

(k) (l)

(m) (n)


neuronsDouble cone

photoreceptors

lowast

lowast lowastlowast

lowastlowast

50120583m

WT

WT

WT

CtBP

zpr

-1Cr

x

1000nmATGmo (333)

ATGmo (375)

ATG

mo

ATGmo

NN

(m998400) (n998400)





OLM

OPL

ZO-1

WT

(a)

ZO-1

ATGmo

(b)

ZO-1

Splice mo

(c)

ZO-1

ATGmo + bves

(d)

aPKC

ONL

(e)

aPKC

(f)

aPKC

(g)

aPKC

(h)

ONL

120573-Catenin

(i)

120573-Catenin

(j)

120573-Catenin

(k)

10120583m 120573-Catenin

(l)







RGC

IPL

INL

OPL

ONL

RPE

Bves knockdown

A loss of normal

retinal lamination

Photoreceptor

defect




4 Discussion











Acknowledgments


References







































In In

(a) (b) (c)

(d)

(e)

(g) (h)

(i) (j) (k) (l)

(f)

WT

IPLONL

OPL

IPL

04

03

02

01

001

(dpf)


WT-lensATGmo-lens

2 3

lowast

lowast

lowast

lowastlowast

lowast

Leng

th (m

m)


WT 3dpf

WT 4dpf

WT 1dpf

ATGmo 3dpf

ATGmo 4dpf







Wild

type

Zn8

Zn8

CALL

CALL

TH

TH

Zpr-1

Zpr-1

Ganglion neurons

(a)

(e) (f) (g) (h)

(b) (c) (d)

(i) (j)

(k) (l)

(m) (n)


neuronsDouble cone

photoreceptors

lowast

lowast lowastlowast

lowastlowast

50120583m

WT

WT

WT

CtBP

zpr

-1Cr

x

1000nmATGmo (333)

ATGmo (375)

ATG

mo

ATGmo

NN

(m998400) (n998400)





OLM

OPL

ZO-1

WT

(a)

ZO-1

ATGmo

(b)

ZO-1

Splice mo

(c)

ZO-1

ATGmo + bves

(d)

aPKC

ONL

(e)

aPKC

(f)

aPKC

(g)

aPKC

(h)

ONL

120573-Catenin

(i)

120573-Catenin

(j)

120573-Catenin

(k)

10120583m 120573-Catenin

(l)







RGC

IPL

INL

OPL

ONL

RPE

Bves knockdown

A loss of normal

retinal lamination

Photoreceptor

defect




4 Discussion











Acknowledgments


References







































Wild

type

Zn8

Zn8

CALL

CALL

TH

TH

Zpr-1

Zpr-1

Ganglion neurons

(a)

(e) (f) (g) (h)

(b) (c) (d)

(i) (j)

(k) (l)

(m) (n)


neuronsDouble cone

photoreceptors

lowast

lowast lowastlowast

lowastlowast

50120583m

WT

WT

WT

CtBP

zpr

-1Cr

x

1000nmATGmo (333)

ATGmo (375)

ATG

mo

ATGmo

NN

(m998400) (n998400)





OLM

OPL

ZO-1

WT

(a)

ZO-1

ATGmo

(b)

ZO-1

Splice mo

(c)

ZO-1

ATGmo + bves

(d)

aPKC

ONL

(e)

aPKC

(f)

aPKC

(g)

aPKC

(h)

ONL

120573-Catenin

(i)

120573-Catenin

(j)

120573-Catenin

(k)

10120583m 120573-Catenin

(l)







RGC

IPL

INL

OPL

ONL

RPE

Bves knockdown

A loss of normal

retinal lamination

Photoreceptor

defect




4 Discussion











Acknowledgments


References







































OLM

OPL

ZO-1

WT

(a)

ZO-1

ATGmo

(b)

ZO-1

Splice mo

(c)

ZO-1

ATGmo + bves

(d)

aPKC

ONL

(e)

aPKC

(f)

aPKC

(g)

aPKC

(h)

ONL

120573-Catenin

(i)

120573-Catenin

(j)

120573-Catenin

(k)

10120583m 120573-Catenin

(l)







RGC

IPL

INL

OPL

ONL

RPE

Bves knockdown

A loss of normal

retinal lamination

Photoreceptor

defect




4 Discussion











Acknowledgments


References







































RGC

IPL

INL

OPL

ONL

RPE

Bves knockdown

A loss of normal

retinal lamination

Photoreceptor

defect




4 Discussion











Acknowledgments


References












































Acknowledgments


References






































knockdown of zebrafish blood vessel epicardial substance results in incomplete retinal lamination

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