Development 110, 325-330 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

325

Localization of specific mRNAs in Xenopus embryos by whole-mount in

situ hybridization

ALI HEMMATI-BRIVANLOU1, DALE FRANK1, MARGARET E. BOLCE1, BOB D. BROWN1, HAZEL

L. SIVE2 and RICHARD M. HARLAND1*

'Department of Molecular and Cellular Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA94720, USA2Department of Genetics, Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washington 98104, USA

* Corresponding author

Summary

We have adapted a non-radioactive technique to detectlocalized mRNAs in whole-mount Xenopus embryos.Synthetic antisense RNA transcribed in the presence ofdigoxygenin-UTP is used as a probe and is detected viaan anti-digoxygenin antibody. We show that localizedmRNAs can be detected from late gastrula to tadpolestages and that high as well as low abundance RNAs canbe detected. The method was tested on muscle actin anda-globin RNAs, whose localization has previously been

characterized. In addition, we used the method todetermine the distribution of XA-1 RNA, an anteriorectoderm-specific RNA, which we show is expressed inthe periphery of the cement gland as well as in the regionof the hatching gland. The sequence of an XA-1 cDNApredicts a protein rich in proline and histidine.

Key words: in situ hybridization, Xenopus, muscle actin,globin, cement gland, hatching gland.

Introduction

Three techniques have been used to analyze localizedgene expression in Xenopus embryos. Since theembryos are large, microdissected tissues can beassayed biochemically for the presence of specificRNAs (Mohun etal. 1984); skilled dissection can yield ahigh degree of spatial resolution (Hopwood et al.19896) but cannot provide information at the level ofsingle cells. Protein products can be localized byimmunological methods, and analysis of embryos bywhole-mount immunohistochemistry provides bothhigh resolution and three-dimensional information(Dent et al. 1989; Hemmati-Brivanlou and Harland,1989); however, after isolating a gene of interest it takesconsiderable time and effort to raise and purify specificantibodies. In situ hybridization is a powerful techniquefor examining the spatial expression of RNAs inembryos, but in situ hybridization to sectioned Xenopusembryos is laborious and not reproducible. The methodhas not been sufficiently sensitive to detect raretranscripts, such as those from many homeobox genes,so that only moderately abundant RNAs have beenanalyzed. Even then an exposure time of weeks (Weeksand Melton, 1987; Sato and Sargent, 1989) or evenmonths (Ruiz i Altaba and Melton, 1989) is oftennecessary. Since in situ hybridization has been carriedout on sectioned tissue, two-dimensional information

must be reconstituted'into three dimensions; this mustbe done either by the imagination of the investigator orby computer (Wilkinson et al. 1987).

Recently a sensitive, non-radioactive in situ hybridiz-ation method has been developed for the localization ofspecific RNAs in whole-mount Drosophila embryos(Tautz and Pfeifle, 1989). We show that a modificationof this technique can be successfully used to detectlocalized RNAs in embryos of the frog Xenopus laevis.In contrast to in situ hybridization of radiolabelledprobes to tissue sections, the non-radioactive method israpid, sensitive and allows staining of whole embryos.

As well as confirming that the method works withgenes such as muscle-specific actin and cr-globin whosetranscripts have previously been localized, we haveanalyzed the expression of the XA-1 gene (Sive et al.1989). From dissection experiments, XA-1 is known tobe expressed in the cement gland and non-brain headectoderm of embryos, but the cellular distribution ofthis transcript has not been determined.

Materials and methods

The in situ hybridization procedure was adapted from Tautzand Pfeifle (1989) and Kintner and Melton (1987).

326 A. Hemmati-Brivanlou and others

EmbryosXenopus laevis were obtained from the Berkeley colonymaintained by the laboratory of J. C. Gerhart. Ovulation offemales and in vitro fertilization were carried out as describedby Condie and Harland (1987). Developmental stages weredetermined according to Nieuwkoop and Faber (1967).

The vitelline membranes of dejellied embryos wereloosened by treatment with proteinase K (5/igml~ ) for 5 tolOmin and were then manually removed. Digestion wasmonitored under the microscope and when the membraneswere seen to lift from the surface the embryos were washed in1/3MR (1XMR=100ITIM NaCl, 1.8mM KC1, lmM MgCl2,2mM CaCl2, 50/igml"1 gentamycin, buffered to pH6.9 with5mM Hepes). Selected embryos were transferred to a 5 mlscrew cap glass vial filled with distilled water. After theembryos settled, the water was removed and the vials werefilled to the brim with MEMFA (0.1M Mops pH7.4, 2mMEGTA, IITIM MgSO4, 3.7% formaldehyde). Embryos wereallowed to fix at room temperature for 1.5 to 2h on aLabquake rotator (Labindustries, Inc). MEMFA was madefreshly from a stock of 10x salts and 37% formaldehyde.Surprisingly, the use of paraformaldehyde at this stage led tohigher background staining. The fix was removed andreplaced with methanol. After a few minutes of equilibrationwith the methanol, the embryos were stored at —20°C.

ProbesThe linearized DNA templates shown in Table 1 were used forin vitro transcription reactions in the presence of digoxigenin-11-UTP (Boehringer Mannheim 1209 256) with either T3, T7or SP6 RNA polymerase (as described by Melton et al. 1985).Linearized template DNA (2.5/ig) was transcribed in 50 jA of40 mM Tris-HCl pH7.9, 6mM MgCl2, 2mM spermidine-HCl,10 min DTT, 1 mM ATP, CTP and GTP, 0.33 mM digoxygenin-11-UTP (BMB 1209 256), 0.66 mM UTP, with 10/iCi of 32P-CTP (400 Ci mmol"1) and 90 units of the appropriate enzymefor 2 to 3h. After standard DNAsel treatment the reactionwas diluted to 100(A. After taking 1/il to determine the totalcounts available, the remainder of the reaction was spunthrough a lml column of Sephadex equilibrated in 0.3 Msodium acetate pH5.5, 0.1% SDS. Following ethanolprecipitation, the RNA pellet was resuspended in 50/il of40 mM sodium bicarbonate, 60 mM sodium carbonate andincubated for 35-50 min at 60°C to generate 200-500nucleotide RNA probes (Lynn et al. 1983). The RNA wasagain precipitated with ethanol and the final pellet wasresuspended in 400 id hybridization buffer; 50% formamide.5xSSC, 500/igmr1 Torula RNA (Calbiochem), 50/igmr1

heparin and 0.1 % Tween 20. A second sample was then takento determine the counts incorporated. The theoreticalmaximum yield (100% incorporation) is 66 /ig.

In situ hybridizationFixed embryos in methanol were gradually rehydrated withME (90% methanol, 10% 0.5 M EGTA) and PTw (lxPBS+0.1% Tween-20) by 5 min incubations in ME, 75%ME+25 % PTw, 50 % ME+50 % PTw, 25 % ME+75 % PTw,100% PTw. They were then washed three times, 5 min each,in PTw. Unless otherwise stated, in all steps of the protocolthe vials were filled almost completely with liquid and rockedon a nutator (Clay Adams). The embryos were then incubatedat room temperature in lOjugml"1 proteinase K (in PTw) onthe nutator. The time of incubation must be adjusted fordifferent batches of proteinase K but 15-30 min has givengood results. Early stage embryos (prior to stage 20) are moresensitive to damage and should be monitored carefully.

Embryos subsequently swell but most stay intact through theprocedure. At the end of the proteinase K treatment,embryos were washed twice, 5 min each, in PTw and wererefixed for 20 min with 4% paraformaldehyde in lxPBS atroom temperature. Paraformaldehyde yielded marginallybetter results than formaldehyde. The refix was followed byfive washes, 5 min each in PTw. Since the embryos aresomewhat delicate after protease treatment, the rocking stepsare done by laying the vials horizontally on the nutator andfilling the vials to reduce turbulence. Where solutions aremore precious and volumes are smaller, the vials are placedvertically in a rack on the nutator.

Hybridization buffer (1 ml) was added to a near empty tubeand the embryos allowed to settle and equilibrate for a fewminutes. The hybridization buffer was changed and theembryos were prehybridized for 1 h at 50 °C. The prehybridiz-ation buffer was replaced with fresh hybridization solutioncontaining the probe (5-10 /ig ml"1) and embryos wereincubated overnight at 50°C. At the end of the hybridizationthe probe was saved. We find that the probe can be recycledup to 3 times. Hybridization was also tested at 65 °C with noobvious improvement in background but with loss ofmorphology, particularly in later stage embryos where theendodermal mass enlarged in comparison to the other tissues.

The embryos were brought from hybridization buffer to2 X SSC gradually by 1.5 ml changes of the following solutions:75% hyb+25% 2xSSC, 50% hyb+50% 2xSSC and 25%hyb+75 % 2xSSC each wash for 10min at 37°C in a shakingwaterbath at between 0 to 60 revs min"1. This was followed bytwo washes 20 min in 2xSSC at 37°C. The non-hybridizedexcess RNA was removed by incubation of the embryos in asolution of 2xSSC containing 20/igmr1 RNase A and 10units ml"1 RNAse T! at 37°C for 30min. From 2xSSC theembryos were transferred to 0.2xSSC and washed twice in0.2XSSC for 30min, at 55°C; and stepped back to PTw,5-10min changes of 75% 0.2xSSC+25% PTw, 50%0.2xSSC+50% PTw, 25% 0.2xSSC+75% PTw and 100%PTw.

RNA hybrids were detected by immunohistochemistry in aprocedure similar to the one described previously (Hemmati-Brivanlou and Harland, 1989). The PTw was replaced withPBT (PBS+2mgmr1 BSA+0.1% Triton X-100), and thevials were rocked at room temperature for 15 min. The PBTwas removed and replaced with 500/il of fresh PBT+10%heat-inactivated goat serum (Gibco; the serum is heated at56°C for 30min). This step saturates non-specific immuno-globulin-binding sites. Even though the commercially avail-able digoxigenin antibody is raised in sheep and therefore theoptimum blocking serum should be sheep serum, we foundthat goat serum worked adequately. Vials were rockedvertically at room temperature for lh. This solution wasreplaced with 500 /il fresh PBT+10 % goat serum containing a1/1000 dilution of the affinity-purified sheep anti-digoxigenincoupled to alkaline phosphatase antibody (BMB cat. no. 1093274). Tubes were rocked vertically overnight at 4°C. Toremove excess antibody, the embryos were washed at least 3times 1 h each with PBT at room temperature (filled up vialsand horizontal rocking).

For the chromogenic reaction with alkaline phosphatase,embryos were washed 3 times, 5 min each at room tempera-ture with lOOmM Tris pH9.5, 50mM MgCl2. lOOmM NaCl,0.1 % Tween 20 and 1 mM Levamisol added freshly to inhibitendogenous alkaline phosphatase. The last wash was replacedwith 1 ml of the same solution and 4.5 /il nitro blue tetrazolium(NBT, Sigma; 75mgml~' in 70% dimethylformamide) and3.5 iA 5-bromo-4-chloro-3-indoyl phosphate (BCIP, Sigma;SOmgmF1 in 100% dimethylformamide) were added directly

Whole-mount in situ hybridization 327

to the tube. Embryos were rocked at room temperature (it isnot critical to protect the reaction from light). The colorreaction was visible from 5min to 60min after treatment.When satisfactory signal and background were observed, thesolution was replaced with PBS. After a total of 2 washes withPBS (5min each at room temperature), the specimens weredehydrated for 3min in methanol and mounted in 2:1 benzylbenzoate: benzyl alcohol (BB/B A) for observation. Althoughsatisfactory for intense signals, such as the actin signal shownin Fig. 2A, this medium dissolves less intense stains.Therefore, a limitation to the sensitivity of the method is thesolubility of the stain. The actin signal was photographed after1 month in BB/BA whereas the globin signal faded in oneweek. For epidermal RNAs, the specimens can be mounted inaqueous media, but for deeper stains, such as those in thenervous system, the embryos must be cleared. If thebackground associated with a particular probe is notsubstantial, the NBT/BCIP reaction can be allowed toproceed for 6 h; under these conditions the blue stain remainshighly localized and is sufficiently intense to remain insoluble.To circumvent the solubility problem, we have also tested theVectastain II kit as suggested by Dent et al. (1989). This kitgives satisfactory results, though it is not as sensitive asNBT/BCIP and the contrast is inferior. HRP-conjugatedsecondary antibodies were also tested and are relativelyinsensitive; however, in cases where mRNA is abundant andNBT/BCIP stains too intensely, the HRP-conjugated anti-body is a useful alternative.

In general we have used albino embryos in theseexperiments. However, the blue stain contrasts well withpigment so that the procedure can be carried out onpigmented embryos (see Fig. 2). We have not attempted tostain embryos bleached with hydrogen peroxide (Dent et al.1989), nor have we tested whether the blue stain is resistant tobleach after the staining is complete.

Northwestern BlotRadiolabelled and dig-11 UTP labelled synthetic RNA wasfractionated by electrophoresis in formaldehyde agarose gelsand transferred to a nylon filter. The filters were exposed toX-ray film to estimate the quality and quantity of the syntheticRNA.

The relative efficiencies of incorporation of dig-UTP bySP6, T7 and T3 RNA polymerases were determined asfollows: Sense transcription templates driven by the SP6, T7and T3 promoters and containing the chloramphenicol acetyltransferase gene, (CAT), were prepared. The transcribedsequences were identical except for short polylinker regions.An aliquot of RNA produced from identical transcriptionreactions was quantitated by 32P-CTP incorporation, and10 ng of each was run out on a formaldehyde agarose gel. Anautoradiograph of the gel was scanned with a Hoeferdensitometer and the peaks integrated to determine therelative quantities of RNA loaded per lane.

The incorporation of digoxigenin in each of the bands wasestimated as follows: The filter was washed for 15 min in PBSand then washed in PBT for 30 min at room temperature. Thiswas followed by an incubation in PBT+10% goat serum forone hour. The anti-digoxigenin antibody (BMB) was diluted1:2000 in PBT+10% goat serum and incubated with the filterfor 2h at room temperature. The filter was then washed inPBT for an hour with 4 changes. PBT was replaced by twochanges of 100HIM Tris pH9.5, 50 mM MgQ, 100 min NaCland 0.1% Tween 20 for 15 min. 4.5/ilmP1 of NBT and3.5/il ml"1 BCIP were added to the last wash and the purplecolor resulting from the chromogenic reaction was detectedafter 5 min. The wet filter was then scanned and the peaks

integrated to quantitate each band; the relative efficiency ofdig-UTP incorporation was then determined based on themasses of RNA present in each band. We have not attemptedto determine the absolute number of digoxygenin groups perRNA molecule.

DNA cloning and sequencingAn apparently full-length cDNA clone (800 bp) of XA-1 wasisolated from a stage 33 Xenopus cDNA head library clonedinto lambda Zap (Stratagene) employing a probe previouslyisolated by subtractive cloning (see Sive et al. 1989). Theplasmid contained within the phage (pBStSK-, into which theoriginal insert was cloned) was excised according to themanufacturer.

Unidirectional exonuclease III deletions of the insert wereconstructed according to Henikoff (1987). After size selec-tion, the sense strand of individual clones was sequenced (assingle-stranded DNA) using the reverse primer and the T7Sequenase kit (United States Biochemical Corporation). Theantisense strand was sequenced after subcloning the entireinsert into pBStSK"1", employing primers based on thepreviously sequenced strand.

Sequences were analyzed employing the Genepro programversion 4.2 (Riverside Scientific Enterprises). Genbank andPIR databases were searched for significant homologies (lodscore of at least 100, requiring a match of 10/30 amino acids).

Results and discussion

Comparison of the efficiency of incorporation ofdigoxigenin-UTP into synthetic RNA using T3, T7and SP6 RNA polymeraseMost transcription vectors currently in use containunique RNA polymerase promoters on either side of apolylinker, allowing the use of experimental antisenseprobes and control sense probes for in situ hybridiz-ation. The comparison between the probes is valid onlyif the relative incorporation of dig-UTP by the RNApolymerases used is approximately equal, and thereforeproduces probes of equal specific activities.

We have tested SP6, T3 and T7 RNA polymerases onsimilar templates in identical reactions containing cP2'P-CTP and dig-UTP. 32P-CTP incorporation was used toquantitate the amount of RNA produced. Samples ofeach RNA were run on a gel and blotted onto nylonmembrane. An autoradiograph of the blot is seen inFig. 1A. The digoxigenin in each band was detectedimmunologically and is shown in Fig. IB. The auto-radiograph and the blot were scanned to quantitate therelative amounts of label present, and we find that thedifferent RNA polymerases incorporate dig-UTP withthe following relative efficiencies: if SP6 is defined as1.0, T3 is 1.4, and T7 is 1.7. Thus all polymerasesincorporate the nucleotide with similar efficiencies.Furthermore, the RNA products are full length,showing that the modified nucleotide does not causepremature chain termination.

Detection of muscle actin RNAAs a first test of the method we set out to detect muscle-specific cardiac actin mRNA, which is abundant andhighly localized in the somites and later in the heart

328 A. Hemmati-Brivanlou and others

1 2 3 1 2

A BFig. 1. Comparative efficiency of digoxigenin-UTPincorporation using T3, T7 and SP6 RNA polymerases.(A) Autoradiogram of 32P-CTP labeled CAT RNAtranscripts blotted onto a nylon membrane. The RNApolymerases used were SP6, T3, and T7, lanes 1, 2, and 3,respectively, under identical reaction conditions. (B) Anti-digoxigenin-11-UTP antibody detection of the RNA on thesame blot as shown in (A). The transcripts are just over900 bases in length.

(Mohun et al. 1984; Dworkin-Rastl et al. 1986; Kintnerand Melton, 1987; Hopwood et al. 1989a). The AC100plasmid (Dworkin-Rastl et al. 1986; Kintner andMelton, 1987) was linearized with PvuW (see Table 1)and was transcribed with SP6 RNA polymerase.Fig. 2A shows the pattern of expression of muscle actinmessage in Xenopus embryos from late gastrula to theswimming tadpole stage. The earliest stage in whichspecific staining was observed was the late gastrula,where two bands of staining correspond to thedeveloping somites (Kintner and Melton, 1987). Somedistortion of early embryos often takes place during theprocedure; in this case the dorsal view makes theblastopore resemble the neural groove of later em-bryos, but observation of the embryo in different planesmakes identification as a late gastrula stage unambi-guous.

The detection of muscle actin RNA at this stageallows us to compare the sensitivities of the two in situhybridization methods. Using radiolabelled probes onsections, Kinter and Melton (1987) detected muscleactin at stage 14. Therefore, the two methods appear tobe comparable in sensitivity, detecting 4x10 tran-scripts per embryo. However, where the radiolabelledprobe required a 5 day exposure, the muscle actin stainshown in Fig. 2 was developed in lOmin.

A problem with the staining of these early stages canbe seen in the background staining of the archenteronfloor of the stage 13 embryo. We also see backgroundstaining of the blastocoel roof of earlier embryos (notshown); this kind of background has been notedpreviously by Hopwood et al. (1989a). Nevertheless,the detection of muscle actin RNA at this early stageillustrates the potential of the whole-mount method. Inparticular the dorsal view of the stage 13 embryo(Fig. 2A) shows how extensive the presumptive headregion is, and how far posteriorly somites are forming atthis stage. At later stages the background is usuallylower and in the stage 24 embryo the RNA is onlydetected in the somites. At later stages such as the stage36 embryo shown here (Fig. 2A), the muscle actinmessage is also detected in the heart (Hopwood et al.1989a) and branchiomeric muscle (Dworkin-Rastl et al.1986). These results show that the whole-mount methodis sensitive and has sufficiently low background to applyto other mRNAs. In addition the advantage ofobserving whole embryos, rather than reconstructing animage from sections, is readily apparent.

Detection of a-globin mRNABefore proceeding with RNAs whose localization hasnot been determined precisely, we also tested an<*-globin probe. The a-globin message is a good testcandidate because it is less abundant than actin attailbud stages and it has been shown to be localized to adifferent region of the embryo, the blood islands whichare part of the ventral mesoderm. It is also useful todevelop a sensitive assay for globin RNA that could beused to investigate the formation of ventral mesodermalcell types in explanted tissues. A cDNA encoding aportion of the Xenopus oTl-globin (Widmer et al. 1981)was subcloned into pGEM-blue to produce paGTldB(M. Bolce unpublished). Transcription with T7 RNApolymerase makes an antisense RNA (see Table 1).Fig. 2B shows the pattern of expression of the globintranscript in a Xenopus tadpole (stage 32). In agree-ment with previous observations, we find this mRNA tobe present exclusively in the ventral mesoderm at thisstage of development. The blood islands bifurcateanteriorly and are fused posteriorly. The sense probedone in parallel did not show any specific hybridization(not shown).

Spatial distribution of XA-1The XA-1 gene is expressed in the presumptive cement

Table 1. Summary of plasmids used with the efficiency of synthesis and amounts used in in situ hybridization

DNA templates

SP65-CATT3-CATT7-CATAC 100paGTldBpaGTldBXA-1XAG-1

Linearizedwith

BamHIXbalHindUIPvullHindlllSmaiNollAfotI

RNA polymeraseused

SP6T3T7SP6T7SP677T7

Probe

SenseSenseSense

AntisenseAntisense

SenseAntisenseAntisense

Amountsynthesized (/ig)

122312188.5

12.63.56.3

Concentrationused (/ig/ml)

N.A.N.A.N.A.

185fi?

12.5

References

Harland and Weintraub, 1985Brown Unpub. dataBrown Unpub. dataKintner and Melton, 1987Bolce Unpub. dataBolce Unpub. dataSive et al. 1989Sive et al. 1989

bp

2A

Fig. 2. Detection of specific mRNAs by whole-mount in situ hybridization. (A) Detection of muscle actin at different stagesof Xenopus development. Embryonic stages 13 (dorsal view), 24 and 36 (lateral views) are presented from top to bottom.The blastopore of the stage 13 embryo is arrowed (bp). In addition to somites the heart (h) of the stage 36 embryo isstained. (B) Localization of the globin mRNA in the blood islands of a stage 32 tadpole. The staining is detected on theventral side of the embryo and is bilaterally symmetrical along the anteroposterior axis. (C) Distribution of the XAG-1transcript in the cement gland of a stage 23 embryo. Note that under saturating conditions of staining some non-specificstaining is also detected on the dorsal side of the embryo. (D-F) Localization of the XA-1 transcript. (D) and(E) represent two different views of a stage 23 embryo; (D) lateral view, (E) anterior view. (F) A stage 36 tadpole viewedin the same plane and magnification as (E). All embryos presented in this figure are albinos with the exception of(B) where a pigmented embryo was used. (A,B and C) have been cleared in benzyl-benzoate/benzyl alcohol to see deepstaining; (D,E and F) have not been cleared.

AAG CAA TCC TCG GCA TTT GAA GAG AGA TAG ACG GGA TTT CCA GCC

ATfi TTTMet Phe

AGC CTTSer Leu

CCT CCTPro Pro

CAT GACHis Asp

GGA GCAGly Ala

GGC AGGm y Arq

GGA GTCGly Vnl

TTC TAC GTTPhe Tyr Val

CCA CAA GGGPro Gin Gly

TCT CCA CCCSer Pro Pro

CTG CAC AGGLeu His Arg

TCC TTA CCTSer Leu Pro

CCT AAG AGAPro Lys Ars.

CTT CTGLeu Leu

AAG ACALys Thr

ATG GGCMet Gly

CCA TCGPro Ser

CCA AAAPro Lys

GAT CTTA«p T.»n

CTT GCTLeu Ala

GGA GAAGly Glu

CCT AGCPro Ser

GGT CACGly His

GAA ACCGlu Thr

CAT CATH l « Hill

CTA ATGLeu Met

GAT TCTAsp Ser

CTT CCTLeu Pro

CCT GAGPro Glu

CCC AATPro Asn

GGG AAAr.lv I.va

GCC CAGAla Gin

CCA GTCPro Val

CCA CCTPro Pro

GAG TTCGlu Phe

GAA CCGGlu Pro

GTT GTCV"' V"'

GGC TGGGly Trp

TTC AGGPhe Arg

GTC AGCVal Ser

AGA ACTArg Thr

AGG CATArg M a

CCC ACTPrn Thr

CCT CAT CACPro Hi m m *

ACA GGAThr Gly

TCT TCTSer Ser

TTC AGGPhe Arg

AGG CACAxfl His

GTC CCTVni Prn

AAT ACC CACAsn Thr His

ACT GGA CGTThr Gly Arg

AGG AGA GATArg Aro Awp

AAG TCALya Ser

CCC TTGPro Leu

CTT CATL^u His

GAA GTCGlu Val

CAT GAAHis Glu

CTA CCCLeu Pro

CAT GGGHl.i Gly

CTA CATLeu His

GAA AACGlu Asn

ATA AAAII* Lys

AAA GCTiil Ala

CAC ACAHis Thr

AGA CCTArg Pro

CCT GAGEXQ Glu

GTC CCTVwl Pro

GAC TGTAsp Cya

AAA GGGLys Gly

ACT GGAThr Gly

CAT CAC ACAHIn Hli Thr

AGC CATSer His

AAC TCAAsn Ser

AAG AAGLya Lya

CGA GTA

AAA GTG

CCT CCC CGAPro Pro Arg

AGT GAA GAASer Glu Glu

CAT TAG TAAHis

GAA AAAGlu Lys

CCA GGCPro Gly

AAG AGALys Arg

TTC CACPhe His

CAT TCCHis Ser

CCA AAAPro Ly»

AAC GGCAsn Gly

ACC TCTThr Ser

CAT GGTHis Gly

AGC AATStr Asn

GCC CACAla His

CAG GAAGin Glu

GGA AAAGly Lys

AAT GACAsn Asp

CAA GGAGin Gly

ACA TAA AAA GAA GAA AAT TAA AGA GGA AAC

CAT TGG ATG GAA TTA TTA ATA AGT TAT GGC AAA ACC A»T

ATA ATG AT

90(15)

135(30)

ISO(45)

225(60)

270(75)

315(90)

360(105)

405(120)

450(135)

495(150)

540(165)

585(180)

630(195)

720

734

Fig. 3. Sequence of XA-1. Nucleotide and conceptualprotein sequence of the longest open reading frame areshown. Nucleotides and amino acids (in parentheses) arenumbered at the end of each line. The first ATG, the firststop codon, the poly A addition signal (AATAAA) and a24 amino acid repeated sequence (see text) are underlined.Two potential glycosylation sites are present in thecarboxyl domain, at amino acids 169 (M3S) and 181(NSS). The poly A tail begins immediately after the lastnucleotide shown.

gland and non-brain head ectoderm from late gastrulato post-hatching stages (Sive et al. 1989; Sive et al.1990). XA-1 is an interesting gene in that its expressionmarks the presumptive head region of the late gastrula(stage 12) before any morphological boundary ismanifest (Sive et al. 1989). This gene has also beenuseful in allowing dissection of the hierarchy ofinductions that occurs during anteroposterior axisformation (Sive et al. 1990).

XA-1 encodes a poly(A)+ RNA of approximately800 bases. An apparently full-length cDNA clone wassequenced on both strands (see Materials and methods)(Fig. 3). The longest open reading frame specifies aprotein of 198 residues, MT approximately 22000. Thereare 3 in-frame methionine residues, with the first in themost favorable context for translation initiation (GCCATG T). The projected protein is rather hydrophilic,except for an 18 residue stretch at the amino end. Thismay be a signal peptide, suggesting that XA-1 may be asecreted or integral membrane protein. The protein isvery proline- (15 %) and histidine- (12%) rich, and onthis basis can be further divided into a more proline-richamino domain and a basic carboxyl domain. The highproline content throughout the protein makes it

Whole-mount in situ hybridization 329

unlikely that XA-1 can form long stretches of unbrokenalpha helix. An interesting motif in this gene is a twice-repeated 24 amino acid sequence, beginning at residues73 and 132, with 20/24 identical residues between therepeats. No striking similarities to previously identifiedopen reading frames were apparent (see Materials andmethods).

The localization of XA-1, determined from dissectionand RNA blot analysis was of low resolution. Wetherefore used the whole-mount in situ hybridizationtechnique to localize the transcripts at single-cellresolution. Antisense RNAs were hybridized to em-bryos of different stages, and after visualizing thehybridized RNA with either NBT/BCIP or the vecta-stain II kit, the embryos were cleared for observation.Since no deep staining was apparent, the embryos wereremounted in PBS for observation of the ectodermalstaining. Figs 2D and E show two views of a stage 23embryo. The transcript is present in cells that form aridge starting behind the otic vesicle, extending alongthe dorsal midline, bifurcating in front of the eyes andreaching the cement gland. The periphery of thecement gland expresses XA-1, in contrast to othergenes, which are expressed in the main mass of cementgland tissue (Jamrich and Sato, 1989). Since it waspossible that the peripheral staining of the cement glandwas due to failure of the probe to penetrate cementgland tissue, we carried out a control hybridization withXAG-1 (Sive etal. 1989). XAG-1 is a suitable candidatesince dissection experiments show that it is almostexclusively expressed in the cement gland at stage 23(Sive et al. 1989). The in situ hybridization shown inFig. 2C confirms this, and shows that the whole of thecement gland can be stained intensely after hybridiz-ation. This result, coupled with the detection of actintranscripts in deep tissues, illustrates that the RNAprobes readily penetrate tissues in frog embryos. Apotential limitation to the technique is also illustrated inFig. 2C. In addition to the cement gland, some non-specific staining is seen in the dorsal structures. Fromthe analysis of RNA in dissected animals (Sive et al.1989), it is unlikely that this signal could be due toXAG-1 RNA. Various sense and antisense probes showvariation in the amount of such background, in a probe-dependent way; it may therefore be necessary tooptimize the fragment of DNA used to generate theprobe (as is the case with RNAse protection) in order tominimize background.

The distribution of XA-1 in the head ectoderm of astage 36 embryo is shown in Fig. 2F. As developmentproceeds the continuous lines of cells that expressed thegene at stage 23 (Fig. 2D,E) break up into patches. Theridge of cells in the dorsal midline is still continuous andextends to a point behind the otic vesicle (not shown),but the facial expression has broken up into patches of afew cells. Expression is excluded from the nasal pits.

The distribution of XA-1 RNA in the head ectodermpartially overlaps the distribution of UVS.2 protein(Sato and Sargent, 1989) and tyrosine hydroxylase(Drysdale and Elinson, 1989). Both these markersappear to be expressed in the hatching gland. However,

330 A. Hemmati-Brivanlou and others

XA-1 and UVS.2 are clearly distinct genes, withdifferent temporal distribution and RNA size.

The representation of XA-1 cDNA clones in librariesindicates that the RNA is rare (less than 1 in 105 embryomRNAs; Sive et al. 1989). However, since theexpression is clearly highly localized it is difficult toassess the number of mRNA copies per cell, and hencethe absolute sensitivity of the method. It is however,encouraging to us that rare transcripts such as XA-1 andengrailed (Xenopus En-2, Hemmati-Brivanlou et al.manuscript in preparation) can be detected with thismethod.

In summary, we have developed a sensitive andreliable method for in situ hybridization in wholeXenopus embryos. The successful detection of thelocalized RNAs, muscle actin and a--globin, shows thatthe method is specific and reproducible, and acomparison of our results to in situ detection of themuscle actin gene with a radioactive probe shows thatthe non-radioactive method is as sensitive. As withother methods, there are problems with backgroundstaining, particularly at early stages. Though thebackground currently limits the sensitivity of thetechnique, further efforts will improve the specificity ofstaining. The solubility of the stain in the currentlyavailable mounting media also limits the sensitivity, butthis difficulty also should be overcome with furtherexperimentation. The use of a non-radioactive assayreduces the time required to develop the signal fromweeks to minutes; in addition the visualization of RNAsin whole embryos greatly facilitates the spatial charac-terization of gene expression. Even where detailedexamination of sectioned material is required, section-ing is more easily carried out after the hybridizationprocedure than before it. Finally, the hybridization andstaining can be carried out on batches of embryospermitting the generation of many pieces of infor-mation in one experiment. Thus, the sensitivity,specificity and convenience of this new method make ita powerful and highly useful tool for the detailed studyof the expression of individual RNAs throughoutdevelopment. The same approach should also provesuccessful in other vertebrate embryos.

A.H.B. would like to thank Nipam Patel for his encourage-ment and helpful comments during the early phase of thework. D.F. is a Rothschild postdoctoral fellow. M.E.B. is anN.S.F. predoctoral fellow. H.L.S. is indebted to Pei FengCheng for expert sequencing, and to Bruce Draper for helpwith isolation of a full-length XA-1 cDNA clone. Supportedby an American Cancer Society postdoctoral fellowship and aNIH Molecular Training Program in Cancer Research grantto H.L.S. who also thanks Hal Weintraub for support. Thiswork was supported by a grant from the NIH (GM 42341).

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{Accepted 26 June 1990)