Isolation of the Xenopus homologue of int-1/wingless and expression during neurula stages of early development

Nucleic Acids Research Isolationof the Xenopus homolog ofint-llwinglessand expression during neurula stagesofearly development

Jasprien Noorderneer, Frits Meijlink, PeterVerrijzer+, FransRijsewijkl andOlivier Destree* Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht and 'DivisionofMolecular Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CXAmsterdam, TheNetherlands

ReceivedOctober 20, 1988; Revised and AcceptedDecember2, 1988 Accessionno. X13138

ABSTRACT

WehaveisolatedtheXenopus homolog (Xint-l) of themouseprotooncogeneint-I froma neurula

stage 17 cDNA library. The deduced protein sequenceof Xint-I includes 371 amino acids. The Xint-lprotein ismoresimilartothemammalianint-Iproduct(69%),thantotheDrosophila counter-partofint-1, wingless (50%).Xint-I shares severalcharacteristics of secreted proteins withtheother

int-I homologs: ithasahydrophobic leader, multiple conserved potential N-linked glycosylation sites and isrich incysteine residues. All 23 cysteines are conserved in the three proteins. Xint-1 istransientlyexpressed during theneurula stages ofearly Xenopus development.

INTRODUCTION

Theint-I geneis aproto-oncogene that isactivated in certain mouse mammarytumors

byintegration of the MMTV provirus in the hostgenome(1-3). Transcripts of thisgene are found in mammary tumors, but no expression is detected in normal adult tissues,

except for the testis of sexually mature mice (4). Expression of the int-I gene during

normal development istemporally and spatially regulated. Transcriptsarefound in murine

embryos between day 9 and 14.5; in situhybridization reveals that RNA accumulation isconfinedtocertain regions of the neural plate and its derivatives (5). The murine int-I producthascharacteristicsofasecretoryprotein: it hasahydrophobic leader,four poten-tial glycosylation sites and is rich in cysteine residues (2). Thegene productenters the

secretory pathway and isglycosylated atseveral sites (6). Int-I isextremely conserved between the mouse and man: only 4 ofthe 370 amino acids aredifferent (7).

Astepforward inunraveling the function ofthe int-1 geneinnormaldevelopmentwas

theidentificationof theDrosophila homolog(8). Almost55% of the amino acidsare

con-servedbetweenDrosophila int-I (Dint-i)andmouseint-i.Dint-i also hasahydrophobic leader,potentialglycosylationsites and all 23cysteineresiduesareconserved. These

struc-turalsimilarities suggest homologous functions of thetwoproteins. Dint-I isexpressed during development, buttranscriptsarehardly detectable in adults. Interestingly, Dint-I

turnedout tobe the knownsegmentpolaritygenewingless (wg)(8-10). Expressionstudies

indevelopmentalmutantsshow thatwglDint-I belongstoahierarchicalnetwork ofgenes thatgovern Drosophila development (reviewed in I1).

Thepresent view on the function ofwg/Dint-1 is thatit functions as anextracellular differentiationfactor that contributestothedeveloping fate ofneighboringcellsby affec-tingtheirgeneexpression(12). There isnostrong evidenceonthefunction ofthemouse

int-i gene,butit issurmised,thatmouseint-i also functionsas anextracellular differen-tiation factor (reviewed in 13).

Recently anothermember of the int-I family which shows a high similarity with the int-I gene has been identified in man: 36% of the aminoacid sequence is identical with thatof int-1 (14). Expressionofthisint-I relatedprotein(irp) isnotrestrictedto develop-ment.

We have initiated a study of the int-1 gene inXenopus laevis. Xenopus is among the fewvertebrateorganismsof whichembryoscanbestudiedin veryearlydevelopment by tissue transplantation. Moreover, it has recently been shown that Xenopus offers great opportunities toanalysetheeffectsofmanipulationof geneactivity byinjectingsenseRNA orantisenseRNAofdevelopmentally regulatedgenesintoembryos (15, 16).Considerable knowledge exists withregard tocelllineage descendenceintheXenopusembryoingeneral (17) andspecifically withregard tothe central nervous system(18, 19). We expectthat adetailed study of theXenopusint-I gene, itsexpression and regulation, willyield new

information with regard to vertebrate patternformation, particularly duringneurogenesis. Inthisreport wedescribethe isolation ofacDNAclonecontaining the Xenopushomolog oftheint-

l/wingless

gene.Theamino acidsequenceishighlyconserved betweenXenopus, mouse, manandDrosophila. Comparison of theint-I and irp-sequences reveals several regions of functional interest that are virtually identical inall four genes. The Xenopus int-I gene is transiently expressedduring the formation ofthe central nervous system.

MATERIALS AND METHODS

Fertilization ofXenopus laevis eggs

Frogs were induced to ovulateby injection of 375 unitsPregnyl (Organon). Eggs were

fertilizedinvitro. Development of the fertilizedeggswasallowedtoproceedat

16-23°C

in 25% MMR(20). The embryosweredejelliedusing2% cysteine-HCl pH7.8. Staging of the embryos was carried out according to the normal table (21).

Screening ofcDNA libraries

cDNAlibrariesof stage 17(22)and stage22-24in

XgtlO,

constructedbyDr. D.A. Melton, were screenedwith mouseint-I cDNAprobes. Twoprobes wereused, the 0.6 kb FnuD H-Cla Ifragment from the 5' end andthe 1.5 kb Cla I-BglII fragment of the 3' end of the mouse int-I gene (23). Hybridization with random-primed DNA probes (24) was

carriedout at42°Cin35% formamide, 5 mMEDTA, 1% glycine, 0.9 MNaCl, 50mM sodiumphosphate pH 7.5, 0.1

%

Ficoll, 0.1% polyvinylpyrrolidoneand100

gg/ml

sheared salmon sperm DNA. The filters were washed in 1xSSC, 0.1% SDS at 50°C. Positive clones were selected and rescreened.

DNA sequencing

For sequence analysis restriction fragments were subcloned in pGEM blue (Promega Biotec). Cloning and analysis were according to standard procedures (25). Nucleotide sequences weredetermined by the dideoxy chain termination method (26) using double stranded supercoiled plasmid DNA (27).

RNA isolation andhybridization

Total RNA was isolated as described (28). Embryos or oocytes were homogenized in guanidinium thiocyanateand RNA waspelletedthrough a CsCl cushion. Poly (A)+RNA

wasselectedonanoligo(dT)-column, fractionated by electrophoresis in 0.8% agarose slab gels containing formaldehyde (6.6% v/v) and transfered to

nitrocellulose

fiters.

Hybridiza-tion with random primed DNA was carried out at 42°C in 50%

formamide,

20Mm sodium phosphatepH6.5, 4xSSC, 5xDenhardt's (25), 0.1 % SDS, 2 Mm sodium pyrophosphate, 10% dextran sulphate and 100

14g/ml

sheared salmon sperm DNA. The final wash was in 0.2xSSC, 0.1% SDS at 65°C.

Nucleic Acids Research

50 100

CGAATAACTAGCACGGT GCTGCCATTCTTCATCATCGGGACTGTATrGCCAAGAAGCCCAGGTATCCCCTTGAGACCTGCACCGACCCCGCCGAC

150 200

CC=TGCCTrCCCCACCr r icG CcT =GCTAT GTCTCCAAAGCTA AACcTCAGGATCCrCACTCTCCGGCCTGAAGAC CTG;GGG

MetArgIleLeuThrPheLeuLeuGlyLeuLysThrLeuTrpV

250 300

rrGGCCTTTCTTCTG CCAACACCATCTGCACAAAICTCAGG AACGTGCr =CCTGG

alLeuAlaRheSerASerLeuSerAsnThrIleAlaVaLAsnAsnSerGlyLysTrpTrpGlyIleVaLAsnValAlaSerAlaGlyAsnValLeuProGI 15 350 400 ATCAGATGCTCGGCCTcTCCrITrlU.AGATCCAAGCCrGCAGCEACTrAGCCGGCAGAAACGTEAATTCGCCAGAACCCACGAAIACLTCAGAGC ySerAspAlaArgProValProLeuValLeuAspProSerLeuGlnLeuLeuSerArgG1nLysArgLeuIleArgGlnAsnProGlyIleLeuGInSer 48 450 500 ATAACCCGAGGCCcrACACAGTGCCATCCGACAGrGCAAATGCATTrAGGAACCCACCGGAACnGCCAACcGAACTcGAACCAAGTrrTGGAA IleThrArgG1yLeuHisSerAlaIleArgGluCysLysTrpHisPheArgAsnArgArgTrpAsnCysProThrGlyThrGlyAsnGInValPheGlyL 82 550 600

AGATAATAACAGAGGCrGCAGAGAAACAGCr-l-IG ICCCATCACCAGCTTGAG ACTCATrC TGCrCGCCTGCTCACAAGGTrCCAT ysIleIleAsnArgGlyCysArgGluThrAlaPheValPheAlaIleThrSerAlaGlyValThrHisSerValAlaArgSerCysSerGluGlySerIl 115 650 700 TCACTCTTGCTCATGTGACTACCGCAGAGGrCCTCGAGGTCCAGACGGCACrGGGCGATGCAGGACACAAGTAATrGGCCGGTCATTCGA eGluSerCysSer,CysAspTyrArgArgArgGlyProGlyGlyProAspTrpHisTrpGlyGlyCysSerAspAsnIleGluEPheGlyArgPheIleGly 148 750 800

AGGGAGlrGTGCrCCAGTGAGAGAGGAGTAGAAATACCIGAAATCA:WiCAACAACCAGGCTGGAGTA CAGTCA CAGTGC

ArgGluPheValAspSerSerGluArgGlyArgAspLeuLysTyrLeuValAsnIauHisAsnAsrGlnAlaGlyArgLeuThrValLauThrGluMeetA 182

850 900

GTCAGGAATGCAATGTCAIGGAATcTCACGATCCrG CCTCACGACcTGCTGGATGCGGCrTCCCCCCCGTTCAGrIrOGGATGC GAAGGA rgGlnGluCysLysCysHisGlyMetSerGlySerCysSerLeuArgThrCysTrpMetArgLAuProProPheArgSerValGlyAspAlaLeuLysAs 215

950 1000

TCUGT ATGGACCTCAGTCACAAGCAC CCAC CTCCAGACACCTCACCAGAACGAAAACCCCACA

pArgTheAspGlyAlaSerLysValThrTyrSerAsnAsnGlySerAsnArgTrpGlySerArgSerAspProProHisLeuGluProGluAsnProThr

248

1050 1100

CATGCrCrGCCATCATCCCAGGATCGrGTCTAF GAGAA;TCCCITCrC CACCCTAGrGAAAAGAATGGAACTCC AACCACAGGGCGAA HisAlaTauProSerSerGlnAspLeuValTyrPheGluLysSerProAsnPheCysSerProSerGluLysAsnGlyThrProGlyThrThrGlyArgI 282

1150

TATGTAACAGCACITCATTGGGACAATGGATTGAACITGCrGTGGTA.GA GGATACCGAGTCTGGCrGAAAAAGrCACTGAACGGTCC

1200

:rr leCysAsnSerThrSerLeuGlyLeuAspGlyCysGluLeuLeuCysCysGlyArgGlyTyrArgSerL.euAlaGluLysValThrGluArgCysHisCy 315

1250

CACATIIrAACTGGTG CATGTCrACcrGCrrGAACrCTAAGCTCACAGATTCATGAGrGCTrTGATATCC 1278 sThrPheAsnTrpCysCysHisValThrCysLeuAsnCysThrSerSerGlnIleValHisGluCysLeuEnd

348

Figure1Nucleotide sequence of part of theXenopusint-IcDNAand the aminoacid sequence deduced for the

int-I protein

The 5' noncoding region, the coding region and part of the 3' untranslated region of the isolated cDNA clone areshown.Thelongest open reading frame spans 1113 nucleotides and is precededbythree stop codons in the sameframe. The sixpotential sites for N-linkedglycosylationandthe stop codons in the leaderareindicated by thin underlining and possible sites for cleavageby proteases are indicated by fatunderlining. Anopen

arrowhead marks thepotential signal peptidecleavagesite.

RESULTS

Isolation andnucleotide sequence

of

theXint-J cDNA

Nucleic Acids Research 60 MRILTFLLGLKTLWVLAFSSLSNTIAVNNSGK---WWGIVNVASAGNVLPGSDARPV Xint-i MGLWA*LPSWVST**L-**LTA*PAAL*A*S**R---******I**ST*L*TD*KSLQ- int-l MDISY*FVIC*MALCSG-GSSL*QVEGKQKSGR*RGSM****AK*GEPN*I-T---PIM- Dint-I MNAP**GIW**-*PLLLTWL*PE**S*---**YMRATGGSSR*MCD-NVPG- irp 120 PLVLDPSLQLLSR- LIRQNPGILQSITRGLHSAIRECKWHFRN RWNCPTGTGNQ-V Xint-i -***E********K R* ********H*VSG**Q**V*****Q** ** ****AP*PH-L int-1

-YMDPAIHST*R*K Rs V*D***V*GALVK*ANL**S**QHQ** ** **S*RNFSRGK Dint-I

-**---*----*QR*--*CHRH*DVMRA*SQ*VAEWTA**QHQ**QH****N*LDRDHSL irp

180

--FGKIINR-GCRETAFVFAITSAGVTHSVARSCSEGSIESCSCDY

--****V**-*******I************************T***~

G P---Xint-I

*--- int-I

NL****VD*-*****S*IY*****A****I**A****T****T***SHQSRSPQANHQAG Dint-I

--**RVLL*SS-**S***Y**S****VFAIT*A**Q*EVK*****P 9M*SAKDSK---- irp 240 ---GGPDWHWGGCSDNIEFGRFIGREFVDSSER-GRDLKYLVNLHNNQAGRLTVLTEMRQ Xint-I ---**************D***LF*******G*K-****RF*M*****E***T**FS**** int-I SVA*VR**E********G**FKFS*****TG**-**N*REKM*****E***AH*QA**** Dint-i ---*IF**--*****w*DY*IKFA*A***AK**K*K*ARA*M*****R***KA*KRFLK* irp 300 ECKCHGMSGSCSLRTCWMRLPPFRSVGDALKDRFDGASKVTYSNNGSNRWGSRSD--- Xint-i ***********TV********TL*A***V*R*******R*L*G*R****A-**AE--- int-1 ***********TVK******AN**VI**N**A*****TR*QVT*--*L*A-TNALAPVSP Dint-I ******V****T*****LAMAD**KT**Y*WRKYN**LQ*VMNQD*TGFT-VANE--- irp 360 --- Xint-i --- int-1 NAAGSNSVGSNGLIIPQSGLVYGEEEERMLNNDHMPDILLENSHPISKIHHPNMPSPNSL Dint-i --- irp 420 ---PPHLEPENPTHALPSSQDLVYFEKSPNFCSPSEKNGTPG Xint-1 ---LLR****D*A*KPP*PH************TY*GRL**A* int-i PQAGQRGGRNGRRQGRKHNRYHFQ*N*H**E*KPPG*K****L*P**S**EKNLRQ*IL* Dint-i ---RFKK-*T---KN******N**DY*IRDREA*SL* irp 480 TTGRICNSTSLGLDGCELLCCGRGYRSLAEKVTERCHCTFNWCCHVTCLNCTSSQIVHEC Xint-I *A**A***S*PA************H*TRTQR*****N***H*****S*R***HTRVL*** int-I *H**Q**E****V***G*M*******RDEVV*V***A***H***E*K*KL*RTKKVIYT* Dint-i *A**V**L**R*M*S**VM******DTSHVTRMTK*G*K*H***A*R*QD*LEALD**T* irp 492 L--- Xint-I *--- int-I *--- Dint-I KAPKNADWTTAT irp

Figure2Comparisonof thepredictedamino acidsequencesofthe Xint-1, mouse int- 1 (2), Dint-I/wingless(8) and irp(14)proteins

Horizontal linesare gaps introduced toalign the four proteins. Conserved amino acids are indicated by an asterisk. Potential glycosylation sites areunderlined, potential protease cleavage sites are boxed.

clone contains aXenopus DNA insert ofapproximately 3.4 kb. The size ofthis cDNA

corresponds with that ofthe transcript detected by RNA blotting (see below).

The nucleotide sequence of the 5'untranslated region and the predicted coding region

ofthe

Xint-1

gene are shown in Figure 1. The longest open reading frame, starting at

theATGcodonatposition158, covers 1113nucleotides, encoding a protein of 371 amino

acids(41 kD). The nucleotides surroundingthestart codon match the consensus sequence

forinitiation of translation(29). As three stop codons are located in the same frame more upstream, we assign the ATG at position 158 as the start codon for translation.

Comparisonof thelengthofthe transcriptwiththe isolated cDNA shows that the length

Nucleic Acids Research i~~~~-6 8 11 15 17 20 20 32 A C A ... : .S ..~~~~~~~... .... ...~ ~ ~ ~ .. ... ... .. ... ...

Figure 3Expressionof the Xint-1 geneduring embryonic development

RNAblotanalysiswasperformedontotalorpoly(A)+RNA from differentstagesofdevelopmentofXenopus laevis(21). Fifteenmicrograms oftotal RNA (panelsA/B)or5micrograms ofpoly (A)+RNA (panels C/D)

werelayeredineach lane. Blotswerehybridizedwith randomprimedXint-l cDNA. Bars indicate the size of themarkers(2.0and 4.5kb)andan arrowmarksthe size of thesingleXint-lItranscriptwithanestimatedlength of 3.5 kb. Panel B showsrehybridizationof the filter in A with the randomprimedH3 histone gene ofX.Ilaevis (35).Panel D showsrehybridizationof the filter inC with the randomprimed cytoskeletalactin gene of Xlaevis(36). Characteristics

of

the

predicted

Xint-J

protein

and

similarity

with

mnt-1,

wingless

and

irp

Analogous

to the mouse and

Drosophila

counterparts, the

predicted

Xint-I

protein

has characteristicsofa

secretory

protein:

it hasa

hydrophobic

leader,

is rich in

cysteine

residues

(23

out of371 amino

acids),

contains several

potential

glycosylation

sites and lacks a transmembrane domain

(31).

Using

the

weight

matrix method described in ref.

32,

we found that the most

likely

site for

cleavage

ofthe

signal peptide

is between amino acids

19 and20.

Theaminoacid sequences of the int- 1 and

irp

products

are

highly

conserved between

Xenopus,

mouse, manand

Drosophila:

69% of the amino acids of the Xint- 1

product

are identical inthe murine andhuman

counterparts,

50% in the

wg/Dint-1

product

and40% inthehuman

irp

product.

Inaddition

14,

16 and 15% ofthe aminoacidsinthe

respective

proteins

is

structurally

similar.

Wg/Dint-1I

hasan insert of 85 aminoacids that is absent inthe other

proteins

(see

Figure

2).

All of the

cysteines

in Xint-1 are conserved in the mouseand

Drosophila

int-1

proteins

andthe

irp

product

lacksoneof the

cysteines.

Con-servation is not restricted to

particular regions

of the

coding

sequence, but found over the entire

length

of the

protein. Apart

from the

cysteines,

some

possible

functional sites areconserved in all four

proteins, potentia

Asn-linked

glycosylation

sitesaswellas

potential

protease

cleavage

sites. TheXint-I

protein

has six

potential

N-linked

glycosylation

sites,

three of which are conserved in mouse andone in

Drosophila.

poten-tial sites for

cleavage by

proteases (33). Three ofthese possible protease

cleavage

sites ofthe murine int-I product areconserved in Xenopus, all being located in hydrophilic regionsof theprotein.Although the predicted proteinstructureof theint-I genes,the biosyn-thesis of the mouse int-I

protein (6)

and the

phenotypes

of

wglDint-1

mutants suggest that theint-I

proteins

aresecreted

(34),

themouseint-1

protein

couldnotbe detected on thecell surfaceorinextracellular fluids

(6).

Thismight indicatethat theprotein is rapidly cleaved outside the cell into fragments.

Expression

of

the int-i gene is

temporally

regulated

Wehave examinedthe

expression

ofXint-1 during

embryonic

development

using RNA blot

analysis.

The Xint-1 gene turns out tobe

transiently

expressed duringneurulation. A

single

transcript of about 3.5 kb can be detected in the early neurula (stage 15) and is stillpresent in mid (17) andlate

(20)

neurula stages (Figure 3A). Noexpression was

found in oocytes, eggs, the blastula and gastrulastages, or in the tailbudstage

(32-34)

(Figure

3C). Rehybridization with a Xenopus H3 histone gene probe (Figure 3B) demonstratedthat somewhatdifferent levelsoftotalRNAwereloaded.The level ofhistone H3 mRNAhas been showntobe nearlyconstantduring

early

development,

whilebeing

higher

in oocytes than in

early developmental

stages

(37). Quantification

ofthe histone H3 and Xint-1 mRNA

expression by densitometry

revealedno

significant

differencesin the levelsof

Xint-1

mRNA in the different neurulastages relative tothe amount of H3 mRNA. The

poly

(A)+RNA was rehybridizedwith aXenopus cytoskeletal actin

probe

(Figure 3D).

The

expression

of

cytoskeletal

actin mRNA was undetectable intheearly stages of

development

but there was anincrease in

cytoskeletal

actin mRNA expression in thetailbud stages, in accordance withthetemporalchange describedbefore

(36).

No Xint-I

transcripts

could be detected in the tailbud stage.

DISCUSSION

Little isknown about the function oftheint-1 gene in early vertebrate development. In

Drosophila, wglint-l

is amemberofa networkofregulatory genes thatdirect

develop-ment

(12).

Most

likely wglint-1

isanextracellular factorthat interactswith receptorson

neighboring cells, changing

their

pattern

of geneexpression

(13).

Supportforthishypothesis

comesfromtheobservationthat

wglint-l

isnotcellautonomous (38)andthat expression ofthe

segmentation

gene

engrailed

in adjacent cellsisinfluencedby

wglint-1

expression

(12).

Thetemporal expression

patterns

of the

int-I

genes during early developmentofthe

mouseandXenopusaresimilar. Int-I expressionwasdetected by RNA blotting from day 9to

day

12.5 ofgestation (4). Bymoresensitive in situhybridizationitwasdemonstrated that

int-I transcripts

continuetobe present inasmallsubpopulationofcells until at least 14.5

days

ofgestation (5).Inthisperiod neurulation,somitogenesis and early organogenesis take

place.

Wecouldnotdetectexpression of Xint-1 in eggs, blastula or in gastrula stages. The Xint-Itranscripts arefirstdetected at stage 15 of embryonic development, the early neural fold stage. In stage 20 embryos, when the neural folds are fused and the neural tube isalmost

closed,

the

Xint-1

mRNAis

still

detectable. In stage32-34 embryos

Xint-1

expression,isnolongerfound. This stage of development would becomparable in several aspects with the day 12 mouse embryo (39, 21).

Nucleic Acids Research andXenopus int-1 is found during similar developmental stages.

The structural similaritywithwglint-1 and thefact that Xint-1 and murineint-I are ex-pressed at the time when formation of the neural tube occurs suggest that Xint-1 may be adifferentation factorinvolved in neuraldevelopment. In Xenopus, neurogenesis hasbeen studiedextensively. It isthought thatpredisposition of theectodermas well as induction by extracellular factors, having a positiondependentrole, are involved in neuralinduction (40). The homeobox containing gene XlHbox6 which is transiently expressed during neurulation isimplicatedin thepredispositionofthe ectoderm to form nerve in response toinductionbythe underlying mesoderm (40). TheXint-1 product, having the structure ofanextracellular proteinand beingtransientlyexpressed during the formation of the neural tube, may be involved in establishing specific local differentiation within the neural ectoderm.

ACKNOWLEDGEMENTS

WethankDr. R. Nusse forvaluable discussionsand for providingunpublished informa-tion.WethankDr. D.A. Melton for generously providingthe cDNA libraries, Dr. T.J. Mohun, and

MI.Heideveld

for kindly providing theactinprobe, J.G. Schilthuisfor giving the H3histoneprobe andforhis help with thecomputer analysis, G.C. Noordermeerand E. Cohen for typing the manuscript and Drs. A.J. Durston and S.W. de Laat for com-ments on the manuscript.

*Towhomcorrespondence should be addressed

'Presentaddress: Laboratory for Physiological Chemistry, State University of Utrecht, Vondellaan 24a, 3521 GGUtrecht, The Netherlands

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