Differential expression of AP-2[agr] and AP-2[bgr] in the developing chick retina: Repression of R

12 Pages·1999·0.77 MB·English

DEVELOPMENTALDYNAMICS214:195–206(1999)
a b
Differential Expression of AP-2 and AP-2
in the Developing Chick Retina: Repression
of R-FABP Promoter Activity by AP-2
DWAYNEA. BISGROVE AND ROSELINE GODBOUT*
DepartmentofOncology,UniversityofAlberta,CrossCancerInstitute,Alberta,Canada
ABSTRACT Retinal fatty acid binding pro- nal cell types (photoreceptor, amacrine, horizontal,
tein(R-FABP)istheaviancounterpartofmurine bipolar,ganglion,andinterplexiform)andasingletype
brain FABP implicated in glial cell differentia- ofglialcell,theMu¨llerglialcell.Thesecellsarisefrom
tionandneuronalcellmigration.R-FABPishighly multipotent progenitor cells that are predominantly
expressedintheundifferentiatedretinaandbrain localizedtoaregionofactivemitosiscalledtheventricu-
of chick embryos. We have previously shown by larzone,locatednexttotheretinalpigmentepithelium
invitrostudiesthattheAP-2transcriptionfactor (Feketeetal.,1994).Post-mitoticcellsmigratefromthe
binds to a consensus AP-2 binding site in the ventricularzonetotheirappropriatestrata,eventually
R-FABP promoter region. Based on the expres- resulting in the characteristic lamellar organization
sionpatternofAP-2inthedevelopingretinaand seeninthematureretina.
onmutationalanalysisoftheAP-2bindingsitein The chicken retinal fatty acid binding protein (R-
FABP) gene may play a role in retinal development.
DNAtransfectionexperiments,weproposedthat
BrainFABP,themammalianorthologueofR-FABP,has
AP-2couldbeinvolvedinthedown-regulationof
been implicated in murine brain development, specifi-
R-FABP transcription. Here, we describe the
cally in the establishment and maintenance of the
cDNAisolationoftwomembersoftheAP-2family
expressed in the chick retina, AP-2a and AP-2b. radial glial fiber system guiding neuronal migration
(Fengetal.,1994;Kurtzetal.,1994).R-FABPishighly
WeshowthatR-FABPmRNAandtheAP-2factors
expressedintheprimitiveneuroectodermalcellsofthe
are expressed in mutually exclusive patterns in
the differentiating retina: whereas AP-2a and undifferentiatedchickretinafromED3toED7,aswell
AP-2b are selectively expressed either in ama- as in the developing brain. There is a 50-fold decrease
in transcript levels from ED7 to ED19 in the retina
crine,orinamacrineandhorizontalcells,respec-
(Godbout, 1993; Godbout et al., 1995). R-FABP, ex-
tively,R-FABPmRNAisfoundinMu¨llerglialcells
pressedduringaperiodofactiveneuronalmigrationin
and/or bipolar cells. Furthermore, a decrease in
the retina, may play an analogous role to that of
R-FABP-dependentexpressionisobtaineduponco-
B-FABPinthebrain.
transfection of primary retinal cultures withAP-2
In a previous study of the regulation of the chicken
expression vectors and a CAT reporter construct.
R-FABPgene,wefoundmultiplesitesofprotein–DNA
Theearlyandcell-specificexpressionofAP-2aand interactionwithin205bpof58flankingDNA(Bisgrove
AP-2binthedevelopingretinasuggestaroleforthis
etal.,1997).Oneofthese,located59bpupstreamofthe
transcription factor family in the early steps of
R-FABP transcription initiation site, corresponded to
amacrineandhorizontalcelldifferentiation.Repres-
the consensusAP-2 DNAbinding site, GCCNNNGGC.
sion of the R-FABP gene in these cells may be an
Supershift experiments using antibodies to AP-2, and
important component of their developmental pro-
methylation interference experiments, indicated that
gram.DevDyn1999;214:195–206. r1999Wiley-Liss,Inc. an AP-2-like transcription factor binds to this region.
Surprisingly, mutation of the AP-2 consensus site did
Keywords: retinaldevelopment;retinalfattyacid
notresultinadecreaseinR-FABPtranscriptionactiv-
bindingprotein;AP-2;amacrinecells;
ity upon DNA transfection of ED5 retinal cultures,
DNAtransfection
suggesting that AP-2 might function as a repressor
rather than an activator of R-FABP transcription.
FurthersupportforthishypothesiscamefromWestern
INTRODUCTION
blots showing an increase inAP-2 levels from ED7 to
To better understand the molecular mechanisms of ED16 in the retina, in contrast to R-FABP mRNA
retinal development, we are studying genes whose
expression patterns suggest a role in cell specification
and differentiation. Chick retinal development begins
Grantsponsor:MedicalResearchCouncilofCanada.
shortlyafterformationoftheopticvesicleonembryonic *Correspondenceto:RoselineGodbout,DepartmentofOncology,Univer-
sityofAlberta,CrossCancerInstitute,11560UniversityAvenue,Edmon-
day (ED) 2 and is complete by the time of hatching on
ton,Alberta,CanadaT6G1Z2.E-mail:[email protected]
ED21. The mature retina contains six different neuro- Received16October1998;Accepted7December1998
r1999WILEY-LISS,INC.
196 BISGROVEANDGODBOUT
levels, which undergo a dramatic decrease during this human AP-2b. Chicken and human AP-2b are 98%
time. identicalattheaminoacidlevel(440/449aminoacids).
Three genes encoding members of the AP-2 family Eightofthenineaminoacidsubstitutionsareconserva-
have been isolated in mammals: AP-2a, AP-2b, and tive. Six of the substitutions are located in the N-
AP-2g.Thespatialandtemporalexpressionprofilesof terminalhalfoftheprotein,whichcontainsthetransac-
the AP-2 factors during embryogenesis suggest that tivationdomain.TheDNAbinding/dimerizationdomain
they are important for normal development. Severe containsthreeaminoaciddifferences,atpositions373,
malformations of the face and skull, as well as abnor- 425,and427.Asimilarlevelofidentityisobservedwith
malitiesofthesensoryorgans,resultfrominactivation mouseAP-2b,exceptthatthemurinesequencehasone
oftheAP-2ageneinmouse(Schorleetal.,1996;Zhang lesscodonthaneitherhumanorchickenAP-2b(codon
et al., 1996). Eye development in the AP-2a-null em- 425).
bryosisseverelyperturbed,withmorphologicaldefects ThepredictedmolecularmassofchickenAP-2bis49
apparentintheopticcupsasearlyasE10.5(P.Mitchell, kDa, similar to that of the 437 amino acid chicken
personal communication). AP-2b gene knock-out mice AP-2a, predicted to be 48 kDa. The predicted amino
die one to two days postnatally because of polycystic acid sequences of chicken AP-2a and AP-2b are 71%
kidneydisease(Moseretal.,1997a).Nooculardefects identical(83%similar)overtheirentirelength(Fig.2).
werereportedinthispaper.Recently,DrosophilaAP-2 Theregionofhighestsequenceconservationlieswithin
has been shown to be expressed in distinct regions of the C-terminal half (DNA binding/dimerization do-
the embryonic head including the visual system, sug- main)with84%identicalresidues(94%similar).There
gestingaroleinthedevelopmentanddifferentiationof arealsolongstretchesofconservedresidueswithinthe
thecentralnervoussystem(MongeandMitchell,1998). proline-richN-terminaltransactivationdomain,particu-
The expression pattern of chicken AP-2 in early larlybetweenresidues40–100,whereAP-2aandAP-2b
embryos from stage 5 (ED1) to stage 28 (ED5.5) sug- are83%identical(87%similar).Thisregionwasshown
gests a role in the budding out of facial prominences to be essential for the transactivation function of hu-
andlimbbuds(Shenetal.,1997).Todefinetheroleof manAP-2a(WilliamsandTjian,1991).
theAP-2 transcription factors in retinal development,
wehaveisolatedandsequencedtheAP-2cDNAspres- AP-2aandAP-2bmRNAandProtein
entinanED16chickretinalibrary.Here,weexamine intheDevelopingEmbryo
the cellular distribution of AP-2a and AP-2b in the AP-2aandAP-2btranscriptlevelsinchicktissuesat
developing chick retina by in situ hybridization and differentdevelopmentalstageswereanalyzedbyNorth-
immunofluorescence analyses and show that R-FABP ernblotting(Fig.3A).ProbesspecifictoeitherAP-2aor
mRNAisexcludedfromretinalcellsthatexpressAP-2. AP-2b transcripts were used for this analysis. Three
We also show that overexpression of AP-2 down- main forms of the AP-2a transcript were observed in
regulatesR-FABPpromoteractivity. theretina,ranginginsizefrom2.5to4kilobases(kb).
Four AP-2b transcripts of different sizes were present
RESULTS intheretina(1.8,2.2,3.5,.5kb).LevelsofbothAP-2a
IsolationofAP-2aandAP-2bcDNAs and AP-2b mRNA peaked at ED10 in the retina.
FromChickRetina
TranscriptswerebarelydetectableatED5.5,theearli-
To identify the AP-2 genes expressed in the chick est stage tested. Although at lower levels than in the
retina,wescreenedanED16retinacDNAlibrarywith retina,theAP-2aandAP-2btranscriptpatternsinthe
human EST probes specific for AP-2a and AP-2b, as ED5.5toED10brainweresimilartothoseobservedin
wellaswiththepreviouslyclonedmouseAP-2gcDNA. theretina,withasignificantdecreaseinlevelsatED16.
Three AP-2a cDNA clones, four AP-2b clones, but no AP-2b mRNAwas relatively abundant in the develop-
AP-2g clones, were obtained upon screening 2 3 105 ing kidney, with two to three times higher levels at
bacteriophage. None of the AP-2a cDNAs were full- ED16comparedtoED7.Ofnote,onlythe2.2kbformof
length,basedonsequenceanalysis.Thesequenceofthe AP-2b was expressed in kidney, suggesting that the
coding region was identical to that of the previously three other forms of AP-2b mRNAfound in brain and
described chicken AP-2a (Shen et al., 1997), although retinaareneuraltissue-specific.AP-2bmRNAwasnot
there were a few alterations in the 38 untranslated detected in either heart or liver, while low levels of
region.NoneoftheAP-2aclonesrepresentedAP-2aB,a AP-2a mRNA were observed in heart and liver on
dominant negative alternative splice form of AP-2a overexposedfilms,decreasingfromED5/6toED16.
describedinmouse(Buettneretal.,1993). Next,weanalyzedAP-2proteinlevelsintheED7to
BothstrandsfromoneoftheAP-2bclonescontaining ED16 retina by Western blotting of whole cell lysates.
the entire open reading frame were completely se- The polyclonal AP-2 antibody used for this analysis
quenced.TheportionofchickenAP-2bcDNAencoding recognizesbothAP-2aandAP-2b,basedonitsabilityto
theopenreadingframeis85%and83%identicaltothat immunoreact with either recombinant (our unpub-
of the human and mouse AP-2b cDNAs, respectively. lished results) or in vitro translatedAP-2a andAP-2b
Fig.1showsanalignmentofthepredictedaminoacid (Moseretal.,1997a,Fig.3).LowlevelsofAP-2protein
sequence of chicken AP-2b with that of mouse and were observed at ED7, followed by a rapid increase at
AP-2INTHEDEVELOPINGRETINA 197
Fig.1. Comparisonofthechicken,human,andmouseAP-2bamino murinesequencewasinsertedinordertopreservesequencealignment.
acidsequences.Identicalresiduesareindicatedintheblackboxesand Thefollowingresidueswereconsideredsimilar:G,A,S,T;E,D,Q,N;R,
conservedresiduesintheshadedboxes.Adashatposition425ofthe K,H;V,M,L,I;F,Y,W.
ED8 and ED9, to reach maximal levels at ED9/ED10 sionofAP-2correlatedwithdecreasedlevelsofR-FABP
(Fig.3B).ThereductioninAP-2levelsatED16reflects mRNA (Godbout, 1993; Bisgrove et al., 1997). To di-
the decrease in AP-2 transcript levels between ED10 rectly test the effect of AP-2 on R-FABP promoter
andED16. activity, we co-transfected AP-2 expression constructs
with an R-FABP/CAT reporter construct containing
InhibitionofR-FABP/CATReporterGene 135 bp of R-FABP 58 flanking DNA including the
ExpressionbyAP-2
consensus AP-2 recognition site (Fig. 4A). Four AP-2
Inapreviousstudy,wefoundthatanAP-2-immuno- expression constructs were prepared, with the coding
reactivetranscriptionfactorwasbindingtoaconsensus regions of AP-2a or AP-2b in the sense or anti-sense
AP-2 recognition site within the promoter of the R- orientation.Co-transfectionofeitherAP-2aorAP-2bin
FABPgene(Bisgroveetal.,1997).TransfectionofpCAT the sense orientation decreased CAT activity by 50%
reporter constructs with mutagenized AP-2 binding comparedtotheanti-senseexpressionvectors(Fig.4B)
sites into undifferentiated ED5 retinal cultures (i.e. (AP-2a,P50.01;AP-2b,P50.0002).NodecreaseinCAT
AP-2-negative)didnotproduceasignificantdecreasein activity was observed upon co-transfection of either
CAT activity, suggesting that AP-2 down-regulates pCAT-basic vector or a construct containing a muta-
ratherthanup-regulatesR-FABPexpression.Arepres- genizedAP-2recognitionsite(datanotshown).Overex-
sor role for AP-2 was corroborated by Northern and pressionoftheAP-2expressionvectorswasverifiedby
Westernblotanalyses,indicatingthatincreasedexpres- Western blot analysis of transfected cell lysates (Fig.
198 BISGROVEANDGODBOUT
Fig. 2. Comparison of chicken AP-2a and AP-2b amino acid sequences. The predicted amino acid
sequencesofchickenAP-2aandAP-2bwerealigned,insertingdashestomaximizealignment.Blackboxes
indicateidenticalresidueswhileshadedboxesindicateconservedresidues.Similarresiduesaredefinedin
Fig.1.
4C). Consistency in the amount of transfected DNA positive cells was higher in the ED7 retina, with the
fromexperimenttoexperimentwasmonitoredbySouth- zone of positive cells extending further towards the
ern blot analysis of Hirt DNA(Fig. 4D). These results anteriorretina(Fig.5d,f).Positivecellswereprimarily
indicate thatAP-2a andAP-2b are capable of repress- confined to the inner nuclear layer (INL). The retina
ingR-FABPpromoteractivityinvivo. differentiates from the posterior to the anterior region
andfromtheinnertotheouterlayer.AP-2-positivecells
LocalizationofAP-2a,AP-2b,andR-FABP
inbothED5andED7arethereforelocatedinthemost
TranscriptsintheRetina
differentiated aspect of the retina. In comparison, R-
If AP-2 can down-regulate R-FABP transcription, FABPtranscriptswerewidespreadthroughouttheINL
thenAP-2 positive cells should have reduced levels of aswellasintheventricular(proliferative)zone(VZ)of
R-FABP mRNA. We therefore studied the distribution ED7 retina (Fig. 5e). By ED11, the inner and outer
ofAP-2a,AP-2b,andR-FABPmRNAinthedeveloping plexiform layers have formed, generating the multi-
retina by in situ hybridization. No AP-2 transcripts layered appearance of the mature tissue consisting of
weredetectedintheED3.5(stage21)retina,incontrast threenuclearlayersseparatedbyplexiformlayers.The
to R-FABP transcripts, which were abundantly ex- AP-2atranscriptswerespecificallyfoundinthevitreal
pressed throughout the tissue (data not shown). By (inner)halfoftheINL,whereamacrinecellbodiesare
ED5, AP-2a- and AP-2b-positive cells (purple) were located (Fig. 5g). AP-2b transcripts were found in
scattered throughout the inner neuroblastic layer amacrinecells,aswellasinathinlayerofcellsinthe
(INBL)oftheposteriorretina(Fig.5a,c),whileR-FABP INL along the outer plexiform layer, identified as
mRNAremainedabundantthroughouttheretina(Fig. horizontal cells based on their characteristic position
5b).At ED7, the inner plexiform layer is beginning to withintheretina(Fig.5i).AP-2btranscriptswerealso
formintheposteriorretinaandisnotyetpresentinthe detected in a subset of cells in the ganglion cell layer,
anterior retina. The number of AP-2a- and AP-2b- likelydisplacedamacrinecellsbecauseoftheirlocation
AP-2INTHEDEVELOPINGRETINA 199
Fig. 4. Overexpression of AP-2a and AP-2b represses R-FABP
promoteractivity.A:MapofthepCAT-135plasmidcontainingtheR-FABP
Fig. 3. Northern and Western blot analyses of AP-2a and AP-2b promoterregionfrom2135to111bplinkedtotheCATreportergene.
expression. A: Northern blots were prepared from poly(A)1 RNA ex- TheAP-2consensusbindingsite(indicatedinbold-facetype)islocated
tractedfromretina(ED5.5,7,10,16),brain(ED5.5,7,10,16),heart(ED6, 59bpupstreamofthetranscriptioninitiationsite.B:Co-transfectionof
15), liver (ED5, 16), and kidney (ED7, 16). The filter was sequentially ED5primaryretinalcultureswithAP-2aandAP-2bexpressionconstructs
hybridizedwith32P-labelled:(i)400bpAP-2bcDNA,(ii)400bpAP-2a and pCAT-135. Level of CAT activity obtained upon co-transfection of
cDNA,and(iii)actinDNA.Thefilterwasstrippedaftereachhybridization. sense and anti-sense pcDNA3/AP-2a and pcDNA3/AP-2b expression
The28Sand18SrRNAmarkersareindicatedontheside.TheAP-2aand constructswithpCAT-135wasmeasuredascpm/µgcellextract/min.The
AP-2b transcripts are indicated by the arrowheads. The asterisk indi- resultsshownrepresenttheaverageoffourexperiments.Thestandard
cates residual signal from the AP-2b hybridization. The extra bands deviations are indicated by the error bars. C: Western blot analysis of
obtainedwiththeactinproberepresenttissue-specificactinmRNAs.B: AP-2aandAP-2blevelsinco-transfectedretinalcultures.Retinalculture
WesternblotswerepreparedfromED7,ED8,ED9,ED10,andED16total lysateswereelectrophoresedina10%SDS-PAGEgelandblottedontoa
chickretinaextracts(100µgprotein/lane).Theproteinswereelectropho- nitrocellulose filter. AP-2a and AP-2b were detected using anti-AP-2
reticallyseparatedbySDS-PAGEandtransferredtoanitrocellulosefilter. antibody.Theresultsfromtwoseparateexperimentsareshown,withthe
Thefilterwasincubatedwithpolyclonalanti-humanAP-2antibodyata AP-2constructsinbothsense(lanes1and2)andanti-sense(lanes3and
1:1000dilution.ThemolecularmassofAP-2is,50kDa. 4)orientations.D:SouthernanalysisofBamHI-digestedHirtDNAfrom
co-transfected retinal cultures. The blot was probed with pCAT-Basic,
whichhybridizestoboththepCAT-135constructandtotheexpression
constructs.TheresultsfromtwoexperimentsareshownwithAP-2aand
immediately next to the inner plexiform layer, if not AP-2b in the sense (lanes 1 and 2) and anti-sense (lanes 3 and 4)
slightlywithinit,aspreviouslydescribed(Ge´nis-Ga´lvez orientations.
et al., 1977). In contrast to the AP-2 transcripts,
R-FABP mRNA in the ED11 retina is confined to the
outerhalfoftheINL(Fig.5h).ThenucleiofMu¨llerglial FABP(2) region and an outer AP-2(2)/R-FABP(1) re-
cellsarefoundpredominantlyinthemiddleoftheINL, gion.
whilebipolarcellbodiesareintheouterhalfoftheINL. Inthemousebrain,radialglialcellsexpressB-FABP
The dual in situ hybridization experiments shown in duringgranulecellmigration(Fengetal.,1994;Kurtz
Fig. 5j and 5k demonstrate the mutually exclusive etal.,1994).Mu¨llerglialcellsrepresentatypeofradial
distributionofR-FABP(brown)mRNAandeitherAP-2a glial cell specific to the retina and may serve a role
(Fig.5j)orAP-2b(Fig.5k)(purple)mRNAintheED11 similar to that of brain radial glial cells (Willbold and
retina, dividing the INL into an inner AP-2(1)/R- Layer, 1998). Carbonic anhydrase II (CA-II) has been
200 BISGROVEANDGODBOUT
Fig.5. LocalizationofAP-2a,AP-2bandR-FABPtranscriptsinthe duallyhybridizedwitheitherAP-2a(purple)(j)orAP-2b(purple)(k),and
developing chick retina. Non-radioactive in situ hybridization was per- R-FABP(brown)antisenseRNA.Displacedamacrinecellsareindicated
formedtodeterminewhichcellsinthedevelopingretinaexpressAP-2a, byarrowsandthehorizontalcelllayerbyarrowheads(i,k).Thesections
AP-2b,andR-FABPmRNA.AdjacentsectionsofED5(a–c),ED7(d–f), areorientedwiththeretinalpigmentepitheliumatthebottomandganglion
ED11(g–l)retinawerehybridizedwithDIG-orfluorescein-labelledAP-2a cellsontop.GCL:ganglioncelllayer;INBL:innerneuroblasticlayer;INL:
(a,d,g),AP-2b(c,f,i),R-FABP(b,e,h)andCA-II(l)antisenseRNA.The innernuclearlayer;IPL:innerplexiformlayer;ONBL:outerneuroblastic
signal was detected by alkaline-phosphatase-coupled secondary anti- layer;ONL:outernuclearlayer;OPL:outerplexiformlayer;RPE:retinal
body,generatingapurpleorbrowncolor.Panellwascounterstainedwith pigmentepithelium;VZ:ventricularzone.Photomicrographsweretaken
ethyl green to show the cell layers. Panels j and k represent sections at2003magnificationusingaNikonDiaphot300microscope.
AP-2INTHEDEVELOPINGRETINA 201
Fig.6. ImmunofluorescentanalysisofAP-2inthedevelopingchick Diaphot300microscope.m,n:Confocalmicroscopeimagesrepresenting
retina. Sections from each developmental stage were stained with dualAP-2(green)andsyntaxin(red)immunofluorescentstainingofED16
anti-AP-2antibody(withFITC-conjugatedsecondaryantibody,green)(b, retina are shown at 2003 (m) and 6003 (n) magnification. Confocal
d,f,h,andj)andcounterstainedwiththefluorescentdyeHoescht33258 images were obtained using a Molecular Dynamics Multiprobe 2001
tolabelthenucleiblue(a,c,e,g,andi).Retinalsectionswereprepared microscope.Scalebars,(m)50µmand(n)20µm.NFL:nervefiberlayer;
from:(a,b)ED5;(c,d)ED7;(e,f)ED10;(g,h)ED16;and(i,j)ED19stage GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear
embryos.Theretinallayersareindicatedini,whilethepositivecelllayers layer; OPL: outer plexiform layer; ONL: outer nuclear layer; RPE: retinal
are labeled in j. k,l: ED16 retina stained with Hoescht 33258 (k) and pigmentepithelium;da:displacedamacrinecells;a:amacrinecells;andh:
monoclonal anti-syntaxin (TRITC-labeled secondary antibody, red) (l). horizontalcells.
Photomicrographs were taken at 2003 magnification using a Nikon
202 BISGROVEANDGODBOUT
shown by others to be specifically expressed in the approximately six nuclei in the posterior retina, while
Mu¨llerglialcellsofthedifferentiatedretina(Vardimon AP-2-positivehorizontalcellsspannedonetotwonuclei
etal.,1986).Wecarriedoutinsituhybridizationwitha (Fig.6i,j).
CA-II riboprobe to determine whether CA-II and R- Syntaxin,aproteininvolvedinsynapticvesicledock-
FABPmRNAhavesimilardistributionprofiles.Fig.5l ing, is a marker of amacrine and horizontal cells
shows an ED11 retinal section hybridized to CA-II (Barnstable et al., 1985; Alexiades and Cepko, 1997).
antisense RNA (purple). The section was counter- Immunostaining of an ED16 retinal section with anti-
stainedwithethylgreeninordertovisualizeallthree syntaxinrevealedextensivestainingoftheinnerplexi-
nuclearlayers.CA-IImRNAwasprimarilyfoundinthe formlayer,asexpectedforaproteininvolvedinsynap-
middleoftheINL,theusuallocationofMu¨llerglialcell tic function (Fig. 6k,l). Staining of the outer plexiform
nuclei. As shown in Fig. 5h, R-FABP mRNA is also layer was somewhat weaker. Cytoplasmic staining of
concentrated in the middle of the INL; however, it amacrine and horizontal cells by anti-syntaxin can be
extends throughout the outer half of the INL where observed in the inner nuclear layer. To determine if
bipolarnucleiarelocated,suggestingabroaderdistribu- syntaxin and AP-2 co-localize, we carried out double
tionfortheR-FABPtranscript. immunofluorescentlabeling.Co-localizationofcytoplas-
micsyntaxin(red)andnuclearAP-2(green)tothesame
ImmunofluorescentAnalysisofAP-2 cells can be seen in Fig. 6m,n. These data provide
intheRetina furtherevidencethattheAP-2positivecellsintheINL
To analyze the cellular distribution of AP-2 during areamacrineandhorizontalcells,asexpectedbasedon
retinal development, we performed immunofluores- theirposition.
cenceanalysisofretinalsectionsatvariousdevelopmen-
DISCUSSION
tal stages.As mentioned earlier, the antibody used in
these experiments recognizes both AP-2a and AP-2b. Inrecentyears,considerableprogresshasbeenmade
There were occasional AP-2 positive cells distributed inidentifyinggenesthataredevelopmentallyregulated
alongtheinnerborderoftheED5posteriorretina(Fig. intheretina(Freundetal.,1996).Functionalanalyses
6a,b). As expected, the AP-2 transcription factor was ofsomeofthesegeneshavegeneratedvaluableinforma-
restrictedtothenucleus.AbandofAP-2positivecells, tion regarding the molecular mechanisms controlling
severalnucleideep,wasfoundintheINLborderingthe cell proliferation and cell fate determination in the
inner plexiform layer of ED7 retina (Fig. 6c,d). The developing retina (Fini et al., 1997; Reh and Levine,
AP-2-expressingcellsextendedintotheanteriorretina 1998).Forexample,experimentsbyAustinetal.,(1995)
nearly to the ciliary epithelium, thinning to a single haveshownthatthetransmembranereceptorNotch-1
layerofcellsinthemid-peripheralretina.Adiscontinu- and its ligand Delta affect the growth and differentia-
ous band of nuclei was also found immediately vitreal tion of retinal cells along the ganglion pathway. How-
tothepresumptiveouterplexiformlayer,thelocationof ever, many gaps remain in our understanding of the
horizontal neurons. Expression in this region was cascade of events underlying the retinal maturation
restricted to the posterior third of the retina. The process.
positivecellsintheINLbetweenthetwobandsofAP-2 The elevated levels of R-FABP mRNA in the chick
expression are presumably migrating amacrine cells. retina from ED3 to ED7, followed by a dramatic de-
MigratingcellswouldstillbeexpectedatED7because crease in transcript levels from ED7 to ED19, suggest
amacrineneurogenesiscontinuesuntilED10(Pradaet that the transcription of the R-FABP gene must be
al., 1991). As with the in situ hybridization experi- tightly regulated in the developing embryo (Godbout,
ments,positivecellswerealsoobservedintheganglion 1993).ThepresenceofaconsensusAP-2bindingsitein
celllayerandlikelyrepresentdisplacedamacrinecells. the R-FABP promoter and the binding of an AP-2-
By ED10, there was a greater number of AP-2- immunoreactivenuclearproteintothisregionindicate
expressing amacrine cells, with most, if not all of the thatoneormoremembersoftheAP-2familyarelikely
cellsinthevitrealhalfoftheINLbeingAP-2positive.A involvedintheregulationoftheR-FABPgene(Bisgrove
positive signal was also detected in the INLbordering etal.,1997).Inourearlierstudy,wedemonstratedthe
the outer plexiform layer where horizontal cells are presenceofAP-2mRNAandproteininthechickretina
located (Fig. 6e,f). As well, AP-2 was present in the (Bisgroveetal.,1997).Atthattime,wewerenotableto
putative displaced amacrine cells that line the outer identifywhichAP-2geneswereexpressedintheretina
border of the ganglion cell layer. Similar results were because the different members of theAP-2 family had
obtained at ED16 when the retinal strata are more not been cloned in chicken. The sequence of chicken
defined (Fig. 6g,h). There was a sharp band of expres- AP-2a cDNA has since been reported by Shen et al.
sion approximately 10 cells wide spanning the inner (1997), and here we have cloned AP-2b from a chick
third of the INL. The staining of the horizontal cell retinacDNAlibrary.WenowshowthatboththeAP-2a
layerwasmoreuniformthanatED10,withadepthof andAP-2bgenesareexpressedatelevatedlevelsinthe
three to four cells. The staining of amacrine and chick retina. The temporal profiles of both genes are
horizontal cells at ED19 had condensed into tighter similar,withbarelydetectablelevelsintheundifferen-
layers, with AP-2-positive amacrine cells spanning tiatedED5retinaandadramaticincreaseintranscript
AP-2INTHEDEVELOPINGRETINA 203
andproteinlevelsfromED7toED9/10.Maximallevels tion(Moseretal.,1997a).However,themouseretinais
are observed at ED9/10, when few retinal cells are still undifferentiated when the AP-2b-/- mice die of
proliferatingandthegreatmajorityofcellsarecommit- polycystic kidney disease one to two days after birth.
ted to a specific differentiation pathway (Prada et al., Expressionofamacrineandhorizontalcelldifferentia-
1991).Moseretal.(1997a)didnotdetecteitherAP-2a tionmarkersoccursprimarilyafterbirth(Gru¨n,1982;
orAP-2bmRNAintheundifferentiatedretinaofday15 Cepkoetal.,1996).Aroleinretinaldifferentiationmay
mouseembryosbyinsituhybridization,althoughtran- therefore only be identified if the lifespan of AP-2b-/-
scriptsweredetectedintheadultmouseeyebyRT-PCR micecanbeextended.Alternatively,differentmembers
(Moser et al., 1995). These results suggest a similar oftheAP-2familymaybeabletocompensateforeach
expression profile for the AP-2 factors in mammalian other in some cell types or tissues, and retinal abnor-
andavianretina.Basedontheirpatternsofexpression, malities may not be observed unless multiple AP-2
we propose that the AP-2 factors are required during genesareinactivated.
theearlystepsofretinaldifferentiation. Although no AP-2g cDNA clones were obtained by
TheAP-2aandAP-2btranscriptsarealsoexpressed screening an ED16 chick retina library, Oulad-Abdel-
in the developing brain and kidney, although levels of ghani et al. (1996) detected AP-2g mRNAin the adult
AP-2a mRNA are considerably lower in these tissues mouseretinabyinsituhybridization,specificallyinthe
than in the retina. The AP-2 transcripts are either inner nuclear layer and ganglion cell layer, although
barely detectable in heart and liver (AP-2a) or not thecellsexpressingthistranscriptwerenotidentified.
detectable (AP-2b). Multiple forms of the AP-2a and AP-2g was not expressed in retinal precursor cells of
AP-2b transcripts are observed in retina and brain, day 11.5 mouse embryos, as determined by in situ
likelyrepresentingalternativesplicingeventsorusage hybridization(Chazaudetal.,1996).OurNorthernblot
ofdifferenttranscriptioninitiationsites,aspreviously analysisofchickretinausingmouseAP-2gcDNAasthe
observed for murine AP-2a (Meier et al., 1995). In probe generated a barely detectable signal (data not
contrast, there is only one major form of the AP-2b shown), suggesting that AP-2g is not expressed or
transcript in kidney, suggesting that the additional weaklyexpressedinthechickretina.
forms found in retina and brain may be neural tissue- Examination of the regulation of the AP-2 genes to
specific. determinehowAP-2expressionisdirectedtohorizontal
BothAP-2genesareselectivelyexpressedinsubsets andamacrinecellsmayleadtoinsightintothenature
ofretinalcellsinthechickembryo:AP-2aisrestricted ofthefactorsinvolvedinthedeterminationanddiffer-
to amacrine cells while AP-2b is specifically found in entiation of these two classes of neurons. In some cell
amacrine,horizontal,andputativedisplacedamacrine types, such as P19 embryonal carcinoma and NT2
cells.AP-2aandAP-2bweredetectedintheED5retina teratocarcinoma, AP-2 expression is inducible by reti-
butnotatED3.5.Neurogenesisofamacrineandhorizon- noic acid (Philipp et al., 1994; Lu¨scher et al., 1989).
talcellsbeginsatED3andED4,respectively(Pradaet These cells undergo neuronal differentiation in re-
al., 1991). It is unlikely that AP-2 plays a role in the sponse to retinoic acid. Induction ofAP-2 could there-
specification of these cell lineages because AP-2 ap- forecoupleresponsetoretinoicacidwiththeregulation
pears to be induced shortly after the cells are commit- ofgenesinvolvedinneuronaldifferentiation.Itwillbe
ted to a particular lineage. However, the early and interesting to determine if retinoic acid, previously
cell-specific expression of AP-2a and AP-2b suggest a implicatedinretinaldevelopment(Dra¨gerandMcCaf-
role for AP-2 in determining the initial events associ- fery, 1996), can up-regulate AP-2 in differentiating
atedwithamacrineandhorizontalcelldifferentiation. amacrine and horizontal cells. If retinoic acid partici-
Others have reported the presence of bothAP-2a and pates in this process, it is likely to be in concert with
AP-2b in specific cell types, leading to the hypothesis differentially expressed proteins involved in retinoic
that combinations of AP-2 factors may cooperate to acid metabolism. It is noteworthy that, unlike other
regulatetheexpressionofspecificsetsofgenes(Moser retinalcells,theprocessesofamacrine,horizontal,and
etal.,1997b).Someofthegenesactivatedorrepressed displacedamacrinecellsareallconfinedtotheirneigh-
by the combination of AP-2a and AP-2b in amacrine boringplexiformlayer,providingacommonalityamong
cellsmaythereforebedifferentfromthoseactivatedor these cell types. Transcription factors restricted to
repressedbyAP-2binhorizontalcells. retinalcellswiththispropertymaybeinvolvedinAP-2
Analysis of both AP-2a-null mice and chimeric mice regulation.
indicates a role for AP-2a in several morphogenic Although theAP-2 family has most commonly been
pathwaysincludingeyeformation,limbpatternforma- associated with transcriptional activation (Duan and
tion, and development of the neural tube, head, and Clemmons, 1995; Dyck and Fliegel, 1995), it has also
body(Schorleetal.,1996;Zhangetal.,1996;Nottoliet been shown to repress the transcription of several
al.,1998).Theoculardefectsinthesemicearesosevere genes(Gaubatzetal.,1995;Getmanetal.,1995;Chen
and appear so early that it has not been possible to etal.,1996).IfmembersoftheAP-2familyareinvolved
assesswhetherAP-2aplaysaroleinretinaldifferentia- in the repression of R-FABP transcription, one would
tion.IncontrasttoAP-2ageneknock-outs,AP-2b-null expectR-FABPmRNAtobeexcludedfromcellsexpress-
miceshownoabnormalitiesotherthanrenalmalforma- ingAP-2.NorthernblotanalysisofAP-2a,AP-2b,and
204 BISGROVEANDGODBOUT
R-FABP indicates a negative correlation between AP- nentoftheiroveralltranscriptionalregulationprogram
2a/bandR-FABPmRNAlevelsduringretinaldevelop- in the retina. A number of transcription factors are
ment, with high R-FABP/lowAP-2 in the undifferenti- expressedintheretina;however,littleisknownregard-
ated retina and low R-FABP/high AP-2 in the ingtheirmechanismsofactionandthenatureoftheir
differentiated tissue. Furthermore, in situ hybridiza- target genes. The identification of twoAP-2 transcrip-
tion and immunofluorescent analyses show mutually tionfactorsspecificallyexpressedintheamacrineand
exclusive expression patterns for theAP-2 factors and horizontal cells of the retina, in conjunction with the
R-FABP transcripts starting at ED10. While R-FABP discovery that R-FABPlikely represents a target gene
mRNA is abundantly and widely expressed in the for these transcription factors, provide the first steps
undifferentiated retina from ED3.5 to ED7, the tran- towards the elucidation of one of the molecular path-
script becomes restricted to the outer portion of the waysunderlyingretinaldifferentiation.
innernuclearlayerwithdifferentiation.Thisregionof
EXPERIMENTAL PROCEDURES
the retina contains cell bodies from Mu¨ller glial cells
ScreeningoftheED16ChickRetinacDNA
and from bipolar cells. R-FABP transcripts are ex-
Library
cluded from amacrine and horizontal cells, in contrast
toAP-2aandAP-2b,whichareselectivelyexpressedin Preparation of the cDNA library was as previously
thesecells.R-FABPmRNAisalsoabsentinphotorecep- described (Godbout, 1993), except that the cDNA was
tors and ganglion cells, neither of which expresses produced from ED16 chick retina poly(A)1 RNA. Ap-
AP-2, suggesting that other transcription factors are proximately 2 3 105 bacteriophage were sequentially
involvedinR-FABPrepressioninthesecelltypes. hybridized with the following: (i) a 1.2 kb EcoRI/
In addition to the correlative studies showing that HindIII human AP-2a cDNA fragment (IMAGE EST
R-FABPmRNAisexcludedfromAP-2-expressingcells, cloneID142203,GenomeSystemsInc,St.Louis,MO),
ourDNAtransfectionexperimentsindicatethatAP-2a (ii) a 1.5 kb EcoRI/HindIII AP-2b cDNA fragment
andAP-2bcandirectlyrepressR-FABPpromoteractiv- derivedfromhumanadultretina(IMAGEESTcloneID
ityinvitro.OverexpressionofeitherAP-2aorAP-2bin 362684, Genome Systems Inc) and (iii) mouse AP-2g
primaryretinalculturesresultedina50%decreasein cDNA (a kind gift from R. Buettner, University of
theactivityoftheCATreportergeneunderthecontrol Regensburg Medical School, Germany). Filters were
of the R-FABP promoter containing the AP-2 recogni- washed at 45°C in 0.13 SSC and 0.1% SDS. Positive
tion site. There was no decrease in CAT activity when cloneswerepurifiedbyadditionalroundsofscreening.
eitherAP-2aorAP-2bantisenseexpressionconstructs ThecDNAendsofpositiveclonesweresequencedusing
were used, or when a pCAT construct carrying a an ABI 310 automated sequencer and the sequences
mutagenizedAP-2sitewastested. analyzed using the BLAST program of the National
MurinebrainFABP,expressedinradialglialcells,is Center for Biotechnology Information, NIH. The se-
involved in the establishment of the radial glial fiber quence of the AP-2b cDNAwas generated by sequenc-
systemguidingneuronalmigrationandmayalsoplaya ingbothstrandsofacDNAcontainingtheentirecoding
roleinrelayinginductivesignalsforneuronaldifferen- region. Sequence data from this article have been
tiation(Fengetal.,1994;Kurtzetal.,1994).Similarto depositedwiththeEMBL/GenBankDatalibrariesun-
B-FABP, R-FABP (initially expressed in neuroectoder- derAccessionNo.AF065140.
mal precursor cells and later restricted to Mu¨ller glial
NorthernBlotAnalysis
cells and/or bipolar cells) may be involved in the
formation of a migratory system for retinal neurons. Poly(A)1RNAwasisolatedfromretina,brain,heart,
The production of R-FABPby Mu¨ller glial cells, which liver,andkidneyatthedevelopmentalstagesindicated
span the entire width of the retina, may play a role in inthelegendtoFig.3.
the positioning and differentiation of all neuronal cell ThemRNAswereelectrophoresedina6%formalde-
typesinthistissue. hyde–1.5%agarosegelinMOPSbufferandtransferred
In summary, we have used two approaches to deter- tonitrocellulose.Thefilterwassequentiallyhybridized
mine whether members of the AP-2 family down- to a 400 bp PstI fragment derived from the 38 end of
regulateR-FABPtranscription.First,weidentifiedthe chicken AP-2a cDNA and to a 400 bp PCR-amplified
AP-2 factors that are expressed in the retina and DNA fragment extending from bp 301–700 of chick
demonstrated mutually exclusive expression profiles AP-2bcDNA.Bothprobeswereselectedonthebasisof
fortwomembersofthisfamilyandR-FABPtranscripts. their low level of similarity to other members of the
Second, we showed by DNAtransfection analysis that AP-2family.Filterswerewashedat45°Cin0.13SSC,
overexpression of either AP-2a or AP-2b in primary 0.1% SDS. The filter was hybridized to mouse actin
retinalculturesresultsinadecreasedactivityfromthe cDNA to control for lane to lane variation in mRNA
R-FABP promoter. These data suggest that these two levels.
AP-2 factors can repress R-FABP transcription. Given
WesternBlotAnalysis
thenumberofgenesshowntoberegulatedbyAP-2in
different systems, repression of the R-FABP gene by Chick retinas were dissected at various stages be-
AP-2aandAP-2bprobablyrepresentsonlyonecompo- tweenED5andED19,andwereimmediatelyfrozenin