the rna-binding protein gene,hermes, is expressed at high ... · pdf filecdepartment of...

The RNA-binding protein gene,hermes, is expressed at high levels in thedeveloping heart

Wendy V. Gerbera,b, Tatiana A. Yatskievychc, Parker B. Antinc,Kristen M. Correiad, Ronald A. Conlond, Paul A. Kriega,b,*

aInstitute for Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USAbDepartment of Zoology, University of Texas at Austin, Austin, TX 78712, USA

cDepartment of Nutrition, University of Arizona, Tucson, AZ 857213, USAdDepartment of Orthopedics, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH 44106, USA

Received 9 September 1998; revised version received 14 October 1998; accepted 14 October 1998

Abstract

In a screen for novel sequences expressed during embryonic heart development we have isolated a gene which encodes a putative RNA-binding protein. This protein is a member of one of the largest families of RNA-binding proteins, the RRM (RNA Recognition Motif)family. The gene has been namedhermes(for HEart, RRM Expressed Sequence). The hermes protein is 197-amino acids long and containsa single RRM domain. In situ hybridization analysis indicates thathermesis expressed at highest levels in the myocardium of the heart andto a lesser extent in the ganglion layer of the retina, the pronephros and the epiphysis. Expression ofhermesin each of these tissues beginsat approximately the time of differentiation and is maintained throughout development. Analysis of the RNA expression of thehermesorthologues from chicken and mouse reveals that, likeXenopus, the most prominent tissue of expression is the developing heart. Thesequence and expression pattern ofhermessuggests a role in post-transcriptional regulation of heart development. 1999 Elsevier ScienceIreland Ltd. All rights reserved

Keywords:RNA-binding protein; RNA recognition motif;hermes

1. Introduction

In addition to transcriptional control, an important andoften overlooked aspect of gene regulation occurs at thepost-transcriptional level. Post-transcriptional regulationmay be achieved at numerous steps in the RNA metabolismpathway, including pre-mRNA-processing (capping, spli-cing and polyadenylation), RNA transport, RNA localiza-tion, translational regulation and RNA stability (reviewed inDay and Tuite, 1998).

Post-transcriptional regulation of gene expression ismodulated by specific interactions of proteins with RNAmolecules. RNA-binding proteins generally fall intofamilies based on the presence of well characterized RNA

binding domains such as the KH domain, the RRM and theRGG box (reviewed by Siomi and Dreyfuss, 1997). How-ever, the presence of a particular RNA-binding motif withina protein does not appear to correlate with any specificregulatory mechanism and members of a single class ofRNA-binding protein may be involved in many differentaspects of post-transcriptional RNA regulation. Amongthe largest families of RNA-binding proteins is the RRMclass. The RRM varies between 80–100 amino acids inlength and is present in one to four copies in different pro-teins. A single RRM domain contains two short stretches ofconserved sequence called RNP1 and RNP2, as well as afew highly conserved hydrophobic residues at specific siteswithin the element. The secondary structure of the RRMdomain is highly conserved. The overall structure isb1a1b2b3a2b4, with the RNP 2 and 1 comprisingb1 andb3, respectively (Burd and Dreyfuss, 1994). Crystal struc-ture of the RRM protein, snRNP U1A, shows that the RNP 1

Mechanisms of Development 80 (1999) 77–86

0925-4773/99/$ - see front matter 1999 Elsevier Science Ireland Ltd. All rights reservedPII S0925-4773(98)00195-6

* Corresponding author. Tel.: +1-512-4710862; fax: +1-512-4719651;e-mail: [email protected]

and 2 domains actually contact the RNA (Nagai et al., 1990;Hoffman et al., 1991), while additional binding specificity iscontributed by residues in the loop region betweenb2 andb3 and by the termini of the domain (Scherly et al., 1990;Bentley and Keene, 1991).

The isolation of mutations in RNA-binding proteins indi-cates that post-transcriptional regulation is essential formany aspects of development, from the initial pattern for-mation in the embryo to maintenance of differentiation ofspecific cell types (reviewed in Bandziulis et al., 1989).Specific members of the RRM family are known to playimportant roles in development. TheDrosophila genesexlethal (sxl), for example, is required for normal sexdifferentiation.sxl contains two RRMs and is involved inthe regulation of both alternative splicing and translationalregulation (Boggs et al., 1987; Bashaw and Baker, 1997;Kelley et al., 1997). AnotherDrosophila gene,elav, con-tains three RRMs and is required for differentiation andmaintenance of neurons. Although the precise mechanismof elavaction is not known, it seems likely to be involved inregulation of both alternative splicing and message stability(Antic and Keene, 1997; Good, 1997). A number of verte-brate genes with homology toelavhave been identified andall are expressed in the nervous system, suggesting that theregulatory role of the protein may be conserved (Yao et al.,1993; Good, 1995).While the study of embryonic heartdevelopment has focused on the role of transcription factors,it seems likely that some aspects of heart development areregulated post-transcriptionally (Murphy, 1996). As theresult of a search for novel genes expressed duringXenopusheart development, we have isolated and characterizedhermes, which encodes a putative RNA-binding protein ofthe RRM family. We have also isolatedhermesorthologuesfrom chicken and mouse and have characterized expressionduring early embryonic development. We find thathermesis expressed at high levels in the developing heart of allembryos examined. The timing of expression suggests arole for hermes in post-transcriptional regulation of myo-cardial gene expression.

2. Results

2.1. Isolation and characterization of the hermes sequencefrom Xenopus, chicken and mouse

ThehermescDNA sequence was isolated in a differentialscreen designed to identify transcripts expressed in thedeveloping heart. The nucleotide sequence and derived pro-tein sequence of a full-lengthhermescDNA is presented inFig. 1A. The poly-A stretch at the extreme 3′ end is notpreceded by a standard 5′-AATAAA-3 ′ polyadenylationsignal, but exactly the same 3′ terminal sequence has beenidentified in all five independent cDNA clones examined,indicating that it represents the terminus of a normal tran-script. The first ATG in the sequence is located at nt 318 and

represents the start of a 594 nucleotide open reading frame,encoding a protein of 197 amino acids. This protein containsa single RRM and no other identifiable domains. A BLASTsearch revealed significant similarity to three othersequences in the GenBank database. The first is theDroso-phila gene,couch potato(cpo), mutants of which exhibitperipheral nervous system defects (Bellen et al., 1992).Sequence similarity between hermes and the cpo proteinis limited to the single RRM while the remainder of theprotein sequences are completely divergent. The hermesRRM also shows similarity to theC. elegansgene,mec-8.Worms mutant formec-8show defects in the attachment ofmuscle to the body wall, apparently due to failure of correctprocessing ofunc-52 transcripts (Lundquist et al., 1996).The mec-8 protein contains two RRM domains andsequence similarity to hermes is limited to the second ofthese domains. Finally, hermes shows significant sequenceidentity to a human gene called RBP-MS type 1 (Shima-moto et al., 1996). This gene was characterized because it islocated in a region of DNA close to the Werner syndromelocus, although it is now known that RBP-MS type 1 is notresponsible for the disease (Shimamoto et al., 1996). In thiscase, similarity of hermes to RBP-MS1 extends over theentire length of the protein. An alignment of the RRMdomains from the hermes, cpo, mec-8 and RBP-MS1 pro-teins is presented in Fig. 1B, together with the RRM con-sensus sequence. The most highly conserved residues liewithin the RNP1 and RNP2 domains that contact the targetRNA (Burd and Dreyfuss, 1994), but it is clear from thisalignment that sequence similarity extends over the entirelength of the RRM. These sequences therefore, may repre-sent a distinct subclass of the RRM protein family.

We have also isolatedhermessequences from chickenand mouse and an alignment of the derived proteinsequences fromXenopus, chicken and mouse is presentedin Fig. 2, together with the human RBP-MS1 sequence.The mouse hermes protein is 78% identical to the frogsequence overall while identity within the RRM is over90%. Amino acid identity between chicken and frog pro-teins is 88% across the entire coding region and 97% in theRRM. TheXenopushermes sequence is 76% identical tothe human RBP-MS1 sequence overall. Based on thisdegree of conservation it seems likely that hermes corre-sponds to the frog orthologue of the RBP-MS1 sequence.Apart from the conserved RRM, all hermes proteins share aconserved carboxyl-terminus domain, with 22 out of thefinal 24 residues being perfectly conserved from frog tohuman.

2.2. Analysis of hermes expression during Xenopusdevelopment

Whole mount in situ hybridization analysis ofXenopusembryos shows thathermesis expressed in the developingheart, pronephros, retina and epiphysis. Expression in theheart is first detected in the tailbud embryo (stage 26), in the

78 W.V. Gerber et al. / Mechanisms of Development 80 (1999) 77–86

Fig. 1. Sequence ofXenopus hermescDNA and the derived protein sequence. (A) The full length cDNA clone contains 1718 nucleotides and encodes aprotein of 197 amino acids. The RNA Recognition Motif (RRM) is underlined. (B) Comparison of the RRM domains contained inhermes, RBP MS,couchpotato(cpo) and the first RRM of mec-8. The RNP1 and RNP2 regions are shaded and RRM consensus residues (Burd and Dreyfuss, 1994) are in bold. Adiagram showing the secondary structure domains is aligned with the RRM sequences.

79W.V. Gerber et al. / Mechanisms of Development 80 (1999) 77–86

paired heart primordia, located on either side of the ventralmidline (Fig. 3A,B). This timing and the pattern of expres-sion is identical to that observed forXenopusmyocardialdifferentiation markers such ascardiac troponin Iandmyo-sin light chain 2(Chambers et al., 1994; Drysdale et al.,1994). As development proceeds, the level ofhermestran-script increases significantly and expression can be detectedthrough the entire length of the heart tube (Fig. 3C) and inthe muscular tissue of the outflow tract (data not shown).Transcript levels remain high throughout heart looping andmorphogenesis (Fig. 3D) and appear to be evenly distribu-ted through all subregions of the heart. Overall, the timingand pattern ofhermesexpression in the developing frogheart appears to be identical to that of myocardial differen-tiation markers.

Also at the tailbud stage,hermesexpression is observedin bilateral stripes on the anterior flank of the embryo. Thisdomain of expression corresponds to the condensing epithe-lium that will form the pronephros (Vize et al., 1995). Asdevelopment proceeds, expression extends further poster-iorly, down the flank of the embryo, coincident with con-densation of the duct epithelium of the pronephros (Fig.3C,D). During later development,hermestranscripts persistthroughout the glomus, tubules and the duct of the prone-phros (Fig. 3G).

At the late tailbud stage (approximately stage 34),hermesexpression becomes apparent in the developing eye and theepiphysis. Expression initially appears as a punctate stain-

ing in the retina, concentrated in the dorsal region of the eye(Fig. 3E). As development proceeds, this domain of expres-sion spreads ventrally and by stage 38,hermesis expressedthroughout the retina (Fig. 3D). At about the same time thatexpression is detected in the developing eye,hermestran-scripts also become visible in the epiphysis (Fig. 3D–F),paired groups of light-sensitive neurons that are part ofthe pineal gland (Eagleson and Harris, 1990). Expressionin the retina and the epiphysis is maintained throughoutsubsequent development.

Histological sections show thathermesexpression in theheart is limited to the myocardium (Fig. 3H). Once again,the pattern of expression is apparently identical to that ofmyocardial differentiation markers (Drysdale et al., 1994).At no stage ishermesexpression detected in the endocar-dium. Sections through the developing kidney indicate thathermesis expressed throughout the epithelium of the pro-nephric duct, glomus and tubules (Fig. 3I). In the eye,hermesexpression is limited to the retinal ganglion cells(Fig. 3J) while the bipolar cells and photoreceptors showno detectable expression. InXenopus, the ganglion cells, theinnermost layer of the retina, first begin to differentiate inthe most dorsal regions at approximately stage 34, and dif-ferentiation then proceeds ventrally through the entire celllayer during subsequent development. The appearance ofhermestranscripts in the eye therefore, correlates with thewave of differentiation of retinal ganglion cells as it movesacross the retinal cell layer.

Fig. 2. Amino acid alignment of hermes proteins from different species. The RRM is underlined. Conserved residues are shaded in dark gray and similarresidues are shaded in light gray.


Fig. 3. Whole mount in situ hybridization analysis of embryonichermesexpression inXenopus. (A–D) and (F–G) are whole mount in situs on albinoembryos, (E) is a whole mount in situ on a pigmented embryo. (A) Early tailbud embryo (stage 26/27) showing expression ofhermesin the heart primordia(black arrow) and the pronephric region (white arrow). (B) Ventral view of embryo shown in (A), indicating the paired heart primordia. (C) Stage 28embryo, showing increase inhermesexpression in the heart and pronephros. (D) Stage 36 embryo showing hermes expression in the heart, pronephros, eyeand epiphysis (at the top of head). (E) Stage 34 embryo, showing onset ofhermesexpression in the eye. Note the punctate staining concentrated in thedorsal region of the eye. (F) Dorsal view of a stage 36 embryo showing paired epiphyseal staining on the top of the head. (G) Lateral view of a stage 36embryo showing duct and tubule staining of the pronephros. (H) Section through the heart region of a stage 31 embryo showshermesexpression is limitedto the myocardial layer of the heart and is absent from the endocardium. (I) Section through the anterior pronephros of a stage 35 embryo.hermesisexpressed throughout the epithelium of tubules and glomus of the pronephros. (J) Section through the eye of a stage 36 embryo showshermesexpression inthe ganglion layer, the inner-most cellular layer of the retina. E, endocardium; M, myocardium; L, lens; P, pigmented epithelium; T, tubules; G, glomus; S,somite.


2.3. hermes expression during avian heart formation

During embryonic chick development expression ofcHermesis primarily restricted to the developing heart.Transcripts ofcHermesare first detected at stage 7–8 in acrescent of anterior lateral mesoderm extending anteriorlyfrom the level of the first somite towards the anterior intest-inal portal (data not shown). This original domain of expres-sion corresponds closely to the localization of transcriptsencoding the homeodomain protein cNkx2-5, whichmarks cardiac precursor cells (Schultheiss et al., 1995).By stage 8,cHermesexpression is restricted to the cardio-genic crescent as the heart primordia begin to fuse along theanterior intestinal portal (Fig. 4A). Transcripts are conspicu-ously absent, however, from a medial region of the anteriorintestinal portal. Sagittal sections through the heart-formingregion at stage 8 reveal thatcHermesexpression is confinedto the cardiogenic mesoderm (Fig. 4E). A few hours later(stage 9),cHermesexpression had coalesced towards themidline as the bilateral cardiogenic regions fuse to formthe primitive heart tube (Fig. 4B). Transverse sections con-firm the localization ofcHermestranscripts to the mesoder-mal component of the forming heart (Fig. 4F). Stainingremains high during completion of heart tube formationand initiation of looping (stage 11; Fig. 4C). By stage 12,however,cHermestranscripts become almost undetectablein the outer curvature of the ventricular region but remainhigh in the developing atrial regions (Fig. 4D) and weakerexpression is also observed in the outflow tract at this time.No other tissues in the chick embryo show significanthermes expression during the first 48 h of development.

2.4. hermes expression in the mouse heart

During early mouse development expression ofhermesislimited to the heart and branchial arches. In the earliest stageembryo examined (e8.5), no expression is visible in hearttissue, although low levels of staining are visible in theadjacent branchial arch tissue (data not shown). In thee9.5 embryomhermesis expressed throughout the heartregion, in the atria, ventricles, sinus venous and the outflowtracts (Fig. 5A). As development proceeds, cardiac expres-sion of mhermesbecomes increasingly regionalized, withsignificantly higher expression in the atria relative to theventricles. This differential expression in the differentheart chambers is conspicuous in sections through theheart of the e10.5 mouse embryo (Fig. 5B). Unlike thechick embryo, wherehermesexpression appears to be com-pletely absent from the ventricles in late stage embryos,mhermesexpression is maintained at low levels in themouse ventricle.

2.5. Adult tissue expression

To determine whetherhermes expression is tissue-restricted in the adult, we have examined transcript levels

in a selection of adultXenopustissues using RNase protec-tion analysis (Fig. 6). This analysis shows thathermesisexpressed at highest levels in adult heart and at moderatelevels in the adult kidney. Expression is extremely low, orabsent, in the liver, lungs and skeletal muscle. Although thissurvey of adult tissues is not comprehensive, the resultssuggest that the tissue-restricted pattern ofhermesexpres-sion detected in the embryo is maintained in adult tissuesand thathermesis expressed at highest levels in heart mus-cle.

3. Discussion

The Xenopus hermessequence was isolated in a screenfor sequences expressed in the developing heart. Based onthe presence of the conserved RRM RNA-binding motif, itappears thathermesencodes an RNA-binding protein.Apart from the RRM domain, hermes contains no otherrecognizable sequence elements that may provide insightinto its possible biochemical function. Thehermessequence is closely related to three previously character-ized genes:Drosophila couch potato (cpo), C. elegansmec-8, and the human geneRBP-MS type I. It seems likelythathermesis the orthologue ofRBP-MS, since the proteinsequences are approximately 70% identical over the entirelength of the coding region. This is consistent with thedegree of identity previously determined for orthologousXenopusand human protein sequences (Tonissen andKrieg, 1994). Unfortunately, no information is currentlyavailable on developmental expression of RBP MS1 andso it is not possible to determine whether the genes exhibitsimilar developmental expression patterns. The relation-ship of hermes to cpo and mec-8 is less clear, since theproteins are completely divergent outside of the RRM.Considering the very different embryonic expression pat-terns however, it seems unlikely thathermesrepresents thefunctional equivalent of eithercpoor mec-8. The unusuallyhigh sequence conservation within the RNA-bindingdomains however, suggests that hermes, RBP MS, cpoand mec-8 represent a subfamily of RRM-class proteinsthat may bind to related sequences.

3.1. hermes expression in the developing heart

During early development of frog, avian and mammalianembryos the highest level ofhermesexpression is detectedin the myocardial tissues of the developing heart (Figs. 3–5). InXenopus, cardiac expression ofhermesis first detectedat about the mid-tailbud stage (stage 26), exactly coincidentwith the time of appearance of myocardial differentiationmarkers. Indeed expression ofhermesin the heart is indis-tinguishable from myocardial differentiation marker expres-sion in both timing and pattern. During subsequentdevelopment of theXenopusheart, at least through forma-tion of the fully chambered heart,hermes expression


appears to be maintained evenly throughout all myocardialtissues, including the muscular tissues of the outflow tract.

Expression ofhermes in the developing chick andmouse heart exhibits some interesting differences com-

pared to frog. Initially, hermes is expressed evenlythroughout the myocardial tissues of the chick and mouseheart and, similar to the situation inXenopus, the onset ofhermesexpression occurs at approximately the same time

Fig. 5. (A) In situ localization ofhermesmRNA during heart formation in mouse. e9.5 embryo showing expression localized in the developing heart. (B)Section through heart region of e10.5 embryo. Highest levels of hermes transcripts are detected in the atrial wall with lower levels in the ventricle.A, atrium;V, ventricle.

Fig. 4. In situ localization ofhermesmRNA during heart formation in chick. (A) Stage 8 chick embryo showingcHermesexpression throughout the cardiaccrescent, except along the medial portion of the anterior intestinal portal (arrowhead). Reaction product visible posterior to the youngest somiteis an artifactof embryo folding. (B) Stage 9 embryo;cHermesexpression remains high as cardiac primordia coalesce to form a single heart tube. (C). Stage 10 embryo.(D) Stage 14 embryo;cHermesexpression is reduced in the ventricular region but remains high in the forming atria. Reduced expression is also observed inthe outflow tract. Inset: higher magnification view of heart in (D). (E) Saggital section through embryo in (A).cHermesexpression is restricted to thecardiogenic mesoderm (cm). (F) Transverse section through the stage 9 embryo shown in (B). IntensecHermesexpression is observed in the cardiogenicmesoderm. AIP, anterior intestinal portal; NT, neural tube; CM, cardiogenic mesoderm.


that transcripts encoding myocardial markers appear. How-ever, during later development of both the chick andmouse heart,hermesexpression becomes restricted tosub-regions of the myocardium. In particular,hermesispreferentially expressed in future atrial tissues and in theoutflow tract. The alteration in the pattern ofhermesexpression closely resembles the shift in expression ofthe embryonic splice form of cardiac Troponin T, whichis initially expressed throughout the heart, but is laterdown-regulated in the ventricles (Sabry and Dhoot,1989). At present there is no evidence for chamber specificexpression of myocardial sequences in the frog heart and sothe differences inhermesexpression between frog andother organisms may reflect fundamental differences ingene expression patterns.

3.2. hermes expression in the kidney and eye

Analysis of theXenopusembryo showshermesexpres-sion in the kidney, eye and epiphysis. During early kidneydevelopment,hermestranscripts are seen in the differentiat-ing pronephros, throughout the epithelium of the duct, thetubules and the glomus. This pattern is distinct from otherkidney markers such as WT1 which is restricted to the glo-mus (Carroll and Vize, 1996), orXlim1 and Pax2 whichmark the duct and tubules (Taira et al., 1994, Heller andBrandli, 1997). We are not aware of marker sequences thatdefine the onset of kidney differentiation inXenopus, but thetiming of hermesexpression corresponds to the time atwhich differentiated kidney structures first become visiblehistologically (Carroll et al., in press).

Beginning at stage 34,hermestranscripts are detectedin the developing eye. Expression is restricted to the re-tinal ganglion cell layer (GCL) and is not detected in anyother retinal cell layers. This pattern of expression is ratherunusual and we are aware of only one other gene,Brn-3.0, that has retinal expression limited to the GCL(Hirsch and Harris, 1997a). Other genes such asPax6andXOptx2that are expressed in the retinal ganglion celllayer are also found in other retinal layers (Hirsch andHarris, 1997b; M. Zuber, M. Perron, A. Bang, C. Holtand W. Harris, personal communication).

3.3. Possible functions for hermes

The presence of the RRM domain suggests that hermesfunctions as an RNA-binding protein. At present however,there is insufficient information to ascribe a specific func-tion for hermes in post-transcriptional regulation of RNAmetabolism. The tissues that expresshermesat high levelsin the developing embryo are not closely related in anyobvious manner. For example, the heart and kidney aremesodermal derivatives, while the retina and epiphysis areneural tissues originally derived from ectoderm. The mostclear correlation betweenhermesexpression in these differ-ent tissues is thathermestranscripts appear at approxi-mately the time that differentiation markers are firstexpressed. This is probably significant, since in additionto alterations in the pattern of gene transcription, differen-tiating cells may also alter their patterns of RNA metabo-lism and processing. Within the RRM, hermes is closelyrelated to theC. elegansmec-8 protein, which is believedto regulate alternative splicing of theunc-52 transcript(Lundquist et al., 1996). The muscle defects in themec-8mutant worm are the result of incorrect splicing of a proteinthat attaches muscles to the body wall. During vertebratemuscle development, a number of genes encoding contrac-tile proteins are spliced differently in cardiac muscle versusother muscle tissues (Cooper and Ordhal, 1984; Sabry andDhoot, 1989; Hardy et al., 1995; Luque et al., 1997).Although we cannot make a direct functional connectionbetween hermes andcardiac troponin Tsplicing in theheart, we note that the localization ofhermesexpressionin the chicken heart correlates with the localization of theembryonic splice variant ofcardiac Troponin T(Sabry andDhoot, 1989). Sincehermesis not expressed in any othermuscle tissues apart from the heart, it is possible that hermesis involved in the processing of cardiac-specific splice var-iants of muscle genes.

4. Materials and methods

4.1. Library screening

Approximately 3× 103 plaques of aXenopus laevisadultheart cDNA library (Ji et al., 1993) were screened at high

Fig. 6. RNase protection analysis ofhermesexpression in adultXenopustissues. Tissue source is indicated at the top of the figure. The position ofthe protectedhermesand Max probes are indicated on the left and sizemarkers on the right.hermestranscripts are abundant in the heart andlower levels of transcript are present in the kidney. Expression is negligiblein the liver, lungs and in skeletal muscle. The ubiquitous Max sequence(Tonissen and Krieg, 1994) is used as a loading control.


stringency with 32P-labeled cDNA probe prepared fromXenopusadult liver RNA and separately with probe contain-ing Xenopusmitochondrial genomic sequences (Rastl andDawid, 1979). The 5′ end sequences of 200 recombinantsnegative for both liver and mitochondrial probes were thendetermined. Genes containing interesting sequence motifswere further characterized by whole mount in situ hybridi-zation. One of these washermes, which contained an RRMdomain. Mouse and chickenhermessequences were iso-lated from an adult mouse heart cDNA library and anembryonic chick cDNA library by low stringency usingXenopus hermescoding region probe.

4.2. In situ hybridization

Digoxygenin-labeled antisense RNA probes were pre-pared using the standard protocol (Boehringer Mannheim).hermestemplate was linearized usingEcoRI and transcribedusing T7 RNA polymerase. Whole mount in situ hybridiza-tion was carried out as described (Hemmati-Brivanlou et al.,1990) with the following modifi-cations: CHAPS wasomitted at all steps, antibody hybridization was in MABbuffer (100 mM maleic acid, 150mM NaCl, pH 7.5) with 2% Boehringer Mannheim blockingreagent. Washes subsequent to antibody hybridiza-tion were in MAB buffer. For sectioning, whole mountstained embryos were dehydrated in ethanol (2× 1 h),incubated in xylene (2× 10 min), 1:1 xylene:paraplast(1 × 10 min), paraplast (1× 30 min, 1× 10–24 h), andthen embedded in paraplast. Twelve-micrometer sectionswere cut and mounted in Permount. Chick embryos werecollected as described in Yatskievych et al. (1997) and fixedin 4% paraformaldehyde in PBS for 2–24 h at 4°C. Follow-ing fixation, embryos were transferred to 12-well plates(Corning Costar Corp.; Cambridge, MA), one embryo perwell, and processed as described according to Nieto et al.(1996). For histology, stained embryos were embedded in7.5% gelatin in 15% sucrose/PBS and frozen in an isopen-tane bath. Embryos were serially sectioned at 20mM andmounted in 95% glycerol. Whole mount in situ hybridiza-tion of mouse embryos was carried as described by Conlon(1996) except that hybridization was at 70°C.

4.3. RNase protection analysis

Total RNA fromXenopusadult male organs was isolatedusing 4 M guanidinium thiocyanate, 25 mM sodium citrate,0.5% sarkosyl, 0.1 Mb-mercaptoethanol. The homogenatewas phenol:chloroform extracted and then isopropanol pre-cipitated. After resuspension in equal volumes of TE and8M LiCl, RNA was recovered by centrifugation and storedas an ethanol precipitate. For RNase protection analysis,hermestranscripts were detected with a32P-labeled anti-sense probe consisting of sequences spanning nt 459–773.Probe for XMax2 was prepared as described previously(Tonissen and Krieg, 1994).

Acknowledgements

Thanks to Eric Olson for providing the mouse heartcDNA library. K.M.C. was supported by a Training Grantfrom the NIH awarded to the Department of Genetics,CWRU. This work was supported by grants, HL54133and HL20220 to P.B.A., an NSF grant to R.A.C. andHL52746 to P.A.K.

References

Antic, D., Keene, J.D., 1997. Embryonic lethal abnormal visual RNA-binding proteins involved in growth, differentiation and posttranscrip-tional gene expression. Am. J. Hum. Genet. 61, 273–278.

Bandziulis, J.R., Swanson, M.S., Dreyfuss, G., 1989. RNA-binding pro-teins as developmental regulators. Genes Dev. 3, 431–437.

Bashaw, G.J., Baker, B.S., 1997. The regulation of theDrosophila msl-2gene reveals a function for sex-lethal in translational control. Cell 89,789–798.

Bellen, H.J., Kooyer, S., D’Evelyn, D., Pearlman, J., 1992. The DrosophilaCouch potato protein is expressed in nuclei of peripheral neuronal pre-cursors and shows homology to RNA-binding proteins. Genes Dev. 6,2125–2136.

Bentley, R.C., Keene, J.D., 1991. Recognition of U1 and U2 small nuclearRNAs can be altered by a 5-amino-acid segment in the U2 small nuclearribonucleoprotein particle (snRNP) B′′ protein and through interactionswith U2 snRNP-A′ protein. Mol. Cell Biol. 11, 1829–1839.

Boggs, R.T., Gregor, P., Idriss, S., Belote, J.M., McKeown, M., 1987.Regulation of sexual differentiation inDrosophila melanogasterviaalternative splicing of RNA from the transformer gene. Cell 50, 739–747.

Burd, C.G., Dreyfuss, G., 1994. Conserved structure and diversity of func-tions of RNA-binding proteins. Science 265, 615–621.

Carroll, T.J., Vize, P.D., 1996. Wilms’ tumor suppressor gene is involvedin the development of disparate kidney forms: evidence from expressionin the Xenopuspronephros. Dev. Dyn. 206, 131–138.

Carroll, T., Wallingford, J., Seufert, D., Vize, P., 1999. Molecular regula-tion of pronephric development. Curr. Top. Dev. Biol. 44, 67–100.

Chambers, A.E., Logan, M., Kotecha, S., Towers, N., Sparrow, D., Mohun,T.J., 1994. The RSRF/MEF2 protein SL1 regulates cardiac muscle-spe-cific transcription of a myosin light-chain gene inXenopusembryos.Genes Dev. 8, 1324–1334.

Conlon, R.A., 1996. Whole mount in situ hybridization to mouse embryos.In: Krieg, P.A. (Ed.), A Laboratory Guide to RNA: Isolation, Analysisand Synthesis, Wiley–Liss, New York, pp. 371-380.

Cooper, T.A., Ordhal, C.P., 1984. A single troponin T gene regulated bydifferent programs in cardiac and skeletal muscle development. Science226, 979–982.

Day, D.A., Tuite, M.F., 1998. Post-transcriptional gene regulatorymechanisms in eukaryotes: an overview. J. Endocrinol. 157, 361–371.

Drysdale, T.A., Tonissen, K.F., Patterson, K.D., Crawford, M.J., Krieg,P.A., 1994. Cardiac troponin I is a heart-specific marker in theXenopusembryo: expression during abnormal heart morphogenesis. Dev. Biol.165, 432–441.

Eagleson, G.W., Harris, W.A., 1990. Mapping the presumptive brainregions in the neural plate ofXenopus laevis. J. Neurobiol. 21, 427–440.

Good, P.J., 1997. The role of elav-like genes, a conserved family encodingRNA-binding proteins in growth and development. Semin. Cell. Dev.Biol. 8, 577–584.

Good, P.J., 1995. A conserved family of elav-like genes in vertebrates.Proc. Natl. Acad. Sci. USA 92, 4557–4561.


Hardy, S., Theze, N., Lepetit, D., Allo, M.R., Thiebaud, P., 1995. TheXenopus laevisTM-4 gene encodes non-muscle and cardiac tropomyo-sin isoforms through alternative splicing. Gene 156, 265–270.

Heller, N., Brandli, A.W., 1997.XenopusPax-2 displays multiple spliceforms during embryogenesis and pronephric kidney development.Mech. Dev. 69, 83–104.

Hemmati-Brivanlou, A., Frank, D., Bolce, M.E., Brown, B.D., Sive, H.L.,Harland, R.M., 1990. Localization of specific mRNAs inXenopusembryos by whole-mount in situ hybridization. Development 110,325–330.

Hirsch, N., Harris, W.A., 1997a.Xenopus Brn-3.0, a POU-domain geneexpressed in the developing retina and tectum. Not regulated by inner-vation. Invest. Opthalmol. Vis. Sci. 38, 960–969.

Hirsch, N., Harris, W.A., 1997b.XenopusPax-6 and retinal development.J. Neurobiol. 32, 45–61.

Hoffman, D.W., Query, C.C., Golden, B.L., White, S.W., Keene, J.D.,1991. RNA-binding domain of the A protein component of the U1small nuclear ribonucleoprotein analyzed by NMR spectroscopy isstructurally similar to ribosomal proteins. Proc. Natl. Acad. Sci. USA88, 2495–2499.

Ji, H., Sandberg, K., Zhang, Y., Catt, K.J., 1993. Molecular sequencingand functional expression of an amphibian angiotensin II receptor. Bio-chem. Biophys. Res. Commun. 194, 756–762.

Kelley, R.L., Wang, J., Bell, L., Kuroda, M.I., 1997. Sex lethal controlsdosage compensation inDrosophila by a non-splicing mechanism.Nature 387, 195–199.

Lundquist, E.A., Herman, R.K., Roglaski, T.M., Mullen, G.P., Moerman,D.G., Shaw, J.E., 1996. Themec-8gene of C. elegans encodes a proteinwith two RNA recognition motifs and regulates alternative splicing ofunc-52transcripts. Development 122, 1601–1610.

Luque, E.A., Spinner, B.J., Dube, S., Dube, D.K., Lemanski, L.F., 1997.Differential expression of a novel isoform of a-tropomyosin in cardiacand skeletal muscle of the Mexican axolotl (Ambystoma mexicanum).Gene 185, 175–180.

Murphy, A.M., 1996. Contractile protein phenotypic variation duringdevelopment. Cardiovasc. Res. 31, 25–33.

Nagai, K., Oubridge, C., Jessen, T.H., Li, J., Evans, P.R., 1990. Crystalstructure of the RNA-binding domain of the U1 small nuclear ribonu-cleoprotein A. Nature 348, 515–520.

Nieto, M.A., Patel, K., Wilkinson, D.G., 1996. In situ hybridization ana-lysis of chick embryogenesis in whole mount and tissue sections. In:Bronner-Fraser, M. (Ed.), Methods in Cell Biology, Vol. 51. AcademicPress, New York.

Rastl, E., Dawid, I.B., 1979. Expression of the mitochondrial genome inXenopuslaevis: a map of transcripts. Cell 18, 501–510.

Sabry, M.A., Dhoot, G.K., 1989. Identification of and changes in theexpression of Troponin T isoforms in the developing avian and mam-malian heart. J. Mol. Cell Cardiol. 21, 85–91.

Scherly, D., Boelens, W., Dathan, N.A., van Venrooj, W.J., Mattai, I.A.,1990. Major determinants of the specificity of interaction between smallnuclear ribonucleoproteins U1A and U2B′′ and their cognate RNAs.Nature 345, 502–506.

Schultheiss, T.M., Xydas, S., Lassar, A.B., 1995. Induction of avian car-diac myogenesis by anterior endoderm. Development 121, 4203–4214.

Shimamoto, A., Kitao, S., Ichikawa, K., Suzuki, N., Yamabe, Y., Imamura,O., Tokutake, Y., Satoh, M., Matsumoto, T., Kuromitsu, J., Kataoka, H.,Sugawara, K., Sugawara, M., Sugimoto, M., Goto, M., Furuichi, Y.,1996. A unique human gene that spans over 230kb in the human chro-mosome 8p11-12 and codes multiple family proteins sharing RNA-bind-ing motifs. Proc. Natl. Acad. Sci. USA 93, 10913–10917.

Siomi, H., Dreyfuss, G., 1997. RNA-binding proteins as regulators of geneexpression. Curr. Opin. Genet. Dev. 7, 345–353.

Taira, M., Otani, H., Jamrich, M., Dawid, I.B., 1994. Expression of theLIM class homeobox geneXlim-1 in pronephros and CNS cell lineagesof Xenopusembryos is affected by retinoic acid and exogastrulation.Development 120, 1525–1536.

Tonissen, K.F., Krieg, P.A., 1994. Analysis of a variantMax sequenceexpressed inXenopus laevis. Oncogene 9, 33–38.

Vize, P.D., Jones, E.A., Pfister, R., 1995. Development of theXenopuspronephric system. Dev. Biol. 171, 531–540.

Yao, K.M., Samson, M.L., Reeves, R., White, K., 1993. Gene elav ofDrosophila melanogaster: a prototype for neuronal specific RNA-bind-ing protein gene family that is conserved in flies and humans. J.Neurobiol. 24, 723–739.

Yatskievych, T.A., Ladd, A.N., Antin, P.B., 1997. Induction of cardiacmyogenesis in avian pregastrula epiblast: the role of the hypoblast andactivin. Development 124, 2561–2570.


the rna-binding protein gene,hermes, is expressed at high ... · pdf filecdepartment of...

Documents