early developmental expression pattern of retinoblastoma tumor suppressor mrna indicates a role in...

Early Developmental Expression Pattern ofRetinoblastoma Tumor Suppressor mRNA Indicates aRole in the Epithelial-to-Mesenchyme Transformation ofEndocardial Cushion CellsMICHAEL WAGNER,* KATHRYN MILES, AND M.A.Q. SIDDIQUICenter for Cardiovascular and Muscle Research, Department of Anatomy and Cell Biology, State University of New YorkHealth Science Center at Brooklyn, Brooklyn, New York 11203

ABSTRACT The earliest stages of embryonicdevelopment are characterized by the generationof precursor cell populations that differentiate andcoalesce into tissue and organ primordia. To pro-vide sufficient numbers of differentiated cells fortissue and organ formation, the differentiative aswell as the proliferative processes of cells must becontrolled and coordinated. Potential regulators ofthe proliferative process include molecules thatcontrol the cell cycle, in particular, the tumor sup-pressor proteins. To begin to understand the rolesuch molecules can play in development, we havestudied the expression of the retinoblastoma tu-mor suppressor (Rb) gene in early chicken devel-opment. Our studies in early chicken embryosshow that Rb is encoded by a single gene that givesrise to several Rb mRNA isoforms through alterna-tive splicing of a primary transcript. These mRNAisoforms potentially encode Rb proteins that differwith respect to the number of sequence motifsknown to target cyclin-dependent kinases to Rb,suggesting dynamic control of Rb phosphorylationand function during development. This complexexpression pattern of Rb mRNA begins as early asthe blastoderm stage of chicken development(stage 3) and continues through stage 18, the lateststage examined. Despite this early embryonic ex-pression of Rb mRNA as detected by reverse tran-scription polymerase chain reaction, Rb mRNAlevels sufficient to be detected by in situ hybridiza-tion were not expressed until after stage 14 of de-velopment. Rb mRNA was found to be localized tocells of the endocardial cushions of the early hearttube, cells of the epicardium, and myogenic cells ofthe somitic myotome. Interestingly, each of thesecell types undergoes an epithelial-to-mesenchymetransformation to form a migratory and/or inva-sive population of mesenchymal cells. We have fo-cused our studies on the expression of Rb mRNA inendocardial cells of the early heart tube, becausethe transition of these cells to mesenchyme ini-tiates the important process of septation, an earlystep in the formation of heart valves.© 2001 Wiley-Liss, Inc.

Key words: retinoblastoma tumor suppressor;cardiac septation; endocardial cush-ion cells; epithelial-to-mesenchymetransformation; epicardium; somiticmyotome; alternative splicing ofRNA; cyclins and cyclin-dependentkinases; hepatocyte growth factor/scatter factor

INTRODUCTION

The earliest stages of vertebrate embryonic develop-ment are characterized by the generation of precursorcell populations from which differentiated cell typesare derived. The proper spatial positioning, controlledexpansion, and subsequent differentiation of these pre-cursor cell populations is critical to the formation oftissue and organ primordia. Although studies of cellmigration, cell signaling, and gene transcription havecontributed much to our knowledge of precursor cellpositioning and differentiation, less is known about themechanisms that control the precise timing and extentof precursor cell expansion to provide populations ofcells for the generation of tissues, organs, and discretestructures within organs.

At the cellular level, proliferation is controlled by theability of the cell to exit the G0 phase of the cell cycleand progress through the G1/S phase restriction pointto enter into the S, G2, and M phases of cell division(Cobrinik et al., 1992, Riley et al., 1994; Weinberg,1995). At the molecular level, progression through thecell cycle is controlled by the complex interplay be-tween several molecules, among which are the cyclins,cyclin-dependent kinases (cdks), and their substrates,i.e., tumor suppressor gene products, in particular, theretinoblastoma tumor suppressor (Rb) (Morgan, 1997).Because mutations of Rb have been associated withtumorigenesis, most studies of Rb function have fo-

Grant sponsor: NIH; Grant number: HL 53573.*Correspondence to: Dr. Michael Wagner, Department of Anatomy

and Cell Biology, SUNY Health Science Center at Brooklyn, 450Clarkson Avenue, Box 5, Brooklyn, NY 11203. E-mail:[email protected]

Received 1 October 2000; Accepted 13 November 2000

DEVELOPMENTAL DYNAMICS 220:198–211 (2001)

© 2001 WILEY-LISS, INC.

cused on its ability to control the cell cycle in normaland neoplastic cells in culture. Few studies have at-tempted to discern whether Rb’s ability to control thecell cycle plays a role in the controlled expansion ofcells to form distinct tissues, structures, and organsduring normal development. To determine whether Rbmight indeed play a role in controlling embryonic cellproliferation and expansion, we have studied the ex-pression of the Rb tumor suppressor gene in earlychicken development, focusing on the heart, which isone of the earliest organ systems to develop.

Heart development involves the precise coordinationof complex morphogenic processes that transform theprimitive heart tube into a four-chambered pump, ableto circulate blood throughout the developing embryo.Achieving this functional subdivision of the primitiveheart and enabling the pump requires the formation ofatrial and ventricular heart chambers, valves to controlthe flow of blood between these chambers, and bloodvessels to convey blood to and from the heart. Thesestructures begin to form when the heart is essentially atube consisting of a myocardium separated from aninner endocardial cell monolayer by an extracellularmatrix called the cardiac jelly. The formation of heartchamber walls, valves, and great vessels begins withthe process of septation in which endocardial cells atdiscrete locations along the heart tube transform intomesenchymal cells that invade and populate the un-derlying cardiac jelly to form “cellularized” outpocket-ings of the cardiac jelly called endocardial cushions.The process of septation begins when the epithelialcells of the endocardium are activated by signals de-rived from the myocardium (Bolender and Markwald,1979; Markwald and Funderburg, 1983). Upon activa-tion, endocardial cells undergo an epithelial-to-mesen-chyme transformation to form the mesenchymal cellsthat migrate into and populate the cardiac jelly to formthe endocardial cushions. Eventually these cells differ-entiate into the fibrous connective tissue of valvularleaflets that form the connective tissue primordia ofheart valves. The establishment of endocardial cush-ions within the embryonic chicken heart tube is anearly step in the formation of the atrioventricularvalves and the aorticopulmonary septum (Eisenbergand Markwald, 1995; van den Hoff et al., 1999).

Our studies show that Rb mRNA is expressed veryearly in chicken embryonic development. In laterstages of development, Rb mRNA attains high levels incells of the endocardial cushions of the heart tube, cellsof the epicardium, the epithelial cell layer surroundingthe myocardium of the heart tube, and cells of thesomitic myotome. These cells represent precursor cellpopulations that expand and differentiate into mesen-chymal cells that eventually contribute to the forma-tion of heart valves, coronary vessels, and skeletalmuscle, respectively. We discuss the role that Rb mayplay in these processes with particular regard to endo-cardial cushion formation.

RESULTS

Isolation of an Rb cDNA From a ChickenEmbryonic Heart cDNA Library and GenomicDNA Analysis

We first sought to isolate and characterize cDNAsencoding tumor suppressors homologous to Rb fromembryonic heart by low stringency screening of achicken 3-day (stage 20) embryonic heart cDNA library(Stratagene) by using a chicken Rb cDNA coding se-quence probe (Feinstein et al., 1994). A single positiveclone, LsRb, containing a 2.25-kbp insert was obtained.Initial restriction site mapping and DNA sequenceanalysis indicated that the structure of this clone dif-fered markedly from that of the chicken 4.7-kbp RbcDNA isolated independently by two laboratories (Boe-hmelt et al., 1994; Feinstein et al., 1994). Comparingthe LsRb 39 sequence starting from the poly A tail withthe 39 end of the published 4.7-kbp Rb cDNA sequenceshowed a block of fully homologous sequence (nucleo-tides 4252 to 4465 in the sequence of Boehmelt et al.,1994) that ended abruptly at nucleotide 4251 followedby nonhomology. A search of Genbank by using theLsRb sequence showed significant homology with thechicken Rb cDNA sequence and no other mRNA se-quences (Boehmelt et al., 1994; Feinstein et al., 1994).This finding indicates that the LsRb cDNA is a homologof the chicken Rb cDNA and that the nonhomologoussequences present in LsRb are not derived from othermRNAs.

The simplest interpretation of these data is that thetwo different cDNAs represent two different mRNAisoforms that arise either as the products of separategenes or as alternatively spliced forms of mRNA tran-scribed from a single gene. To address this, we per-formed a Southern blot analysis of chicken genomicDNA to assess Rb gene copy number. Chicken genomicDNA was digested with restriction enzymes that do notcleave the Rb cDNA and whose cutting would be de-pendent on sites within intronic and/or flanking se-quences. (Over 95% of the 100-kbp human Rb gene isintronic sequence and the exons corresponding to thesequence of the chicken cDNA probe used in this blotare interrupted by 3–4 introns [Lee et al., 1987]). De-spite cutting within the flanking or intron sequences ofthe Rb gene, which are at least 20-fold more complexthan the total exonic sequences and which would pre-sumably give a more complex restriction pattern in thecase of two or more Rb genes owing to genetic drift, therestriction pattern obtained was relatively simple (Fig.1A). Indeed, the pattern observed here is as simple asthat generated by digestion of chicken genomic DNAwith HindIII, an enzyme that cuts within the codingregion of the chicken Rb gene four times (Bernards etal., 1989). Together, these results suggest that as withhumans, chickens seem to have only a single Rb gene(Bernards et al., 1989).

199RETINOBLASTOMA TUMOR SUPPRESSOR MRNA EXPRESSION IN ENDOCARDIAL CUSHION CELLS

Analysis of Rb mRNA Expression DuringDevelopment and in Different Tissues

We next sought to determine whether we could de-tect the expression of multiple Rb mRNAs during de-velopment and within different tissues. By using thechicken LsRb cDNA to probe early embryonic mRNA, asingle major mRNA species was detected that corre-sponded to the previously characterized 4.7-kb Rb

mRNA (Boehmelt et al., 1994; Feinstein et al., 1994)(Fig. 1B). When variation in mRNA loading was ac-counted for, it appeared that the 4.7-kb Rb mRNA ispresent as early as blastoderm stages 3 through 5, withexpression peaking at approximately stages 11 through13. A faint lower molecular weight band running atapproximately 3.0 kb was also apparent in stages 3through 5 mRNA. In Figure 1C, the 4.7-kb Rb mRNA

Fig. 1. A: Southern blot of chicken liver genomic DNA. Blot washybridized with a 32P-labelled 1.6-kbp EcoRI fragment from a chicken Rbcoding region cDNA (kindly provided by Dr. T. Gilmore). Washes were at.23 SCC, 65°C, and the blot was exposed to X-ray film for 20 hr.HindIII-digested lambda DNA served as molecular weight marker. B: De-velopmental RNA blot of total chicken embryo RNA. Poly A1 RNAsamples from staged chicken embryos were subjected to Northern blotanalysis and probed with the 1.6-kbp Rb coding region probe (upper

panel). RNA molecular weight markers are denoted on the left. The lowerpanel shows the hybridization signal from the same blot rehybridized witha chicken ribosomal protein L37a cDNA probe to control for loadingdifferences between lanes. C: Tissue specificity RNA blot. Poly A1 RNAsamples from different tissues of a newborn chicken were subjected toNorthern blot analysis and hybridized with the 1.6-kbp Rb coding regionprobe (upper panel; “Sk” means skeletal). The lower panel is the L37aloading control hybridization.

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and a lower molecular weight Rb-specific band (approx-imately 3.8 kb in size) were present in all tissues ex-amined. Skeletal muscle and liver appeared to expresslow levels of Rb mRNA, whereas neural tissues, asrepresented by hindbrain, cortex, and spinal cord mR-NAs, exhibited high levels of Rb mRNA expression.These data show that at least two Rb mRNA isoforms,the 4.7 and 3.8-kb mRNAs, are expressed from a singlegene in several different tissues.

Reverse Transcription Polymerase ChainReaction Analysis of Rb mRNA ExpressionDuring Development and in Early Heart Tube

Our data suggest that in addition to the reported4.7-kb Rb mRNA, other Rb mRNA isoforms exist thatmay arise from alternative splicing of RNA transcriptsfrom a single gene. That one of these isoforms, LsRb,was obtained from an embryonic chicken heart cDNAlibrary indicates that alternative splicing of Rb mRNAoccurs in early heart tissue and may play an importantrole in regulating the expression and perhaps the func-tion of Rb during chicken development. To address thispossibility, we resorted to the more sensitive method ofreverse transcription polymerase chain reaction (RT-PCR) to determine whether LsRb and other Rb mRNAisoforms were expressed during early stages of chickendevelopment (Hamburger and Hamilton stages 3–15)and whether other Rb mRNA isoforms in addition toLsRb are expressed in early heart tube.

We designed RT-PCR oligonucleotide primers to en-compass the region of the LsRb clone that appeared,when compared with the 4.7-kb Rb cDNA sequence, tohave undergone alternative splicing (see Fig. 3B, BandD arrows). RT-PCR analysis was performed by usingmRNA obtained from embryos at different develop-mental stages and from stages 16–18 heart tube.Stages 16–18 chicken embryos were selected as asource of heart tube mRNA, because heart tubes fromthis stage can be easily dissected away from gut ortrunk tissues that could contaminate heart tube mRNAwith non-heart tube mRNAs. RT-PCR analysis of earlystage total chicken embryo mRNA showed three RbRT-PCR products with the most prominent productrepresented by band B (Fig. 2A). RT-PCR analysis ofstage 16–18 chicken embryo heart tube mRNA showedfive Rb RT-PCR products (Fig. 2B). To verify the au-thenticity of the heart tube RT-PCR products obtainedwith the PCR primers used, we performed a series ofincreasingly stringent PCR reactions by using highertemperatures of annealing. All five Rb RT-PCR prod-ucts are amplified from heart tube mRNA even underthe most stringent annealing temperatures for thesereactions (70°C) (Fig. 2B). Comparing the pattern of RbRT-PCR products between heart tube and total embryoshows that products B, C, and D are amplified fromtotal embryo mRNA as well as from heart tube mRNAsuggesting that the mRNAs from which these RT-PCRproducts are derived are present in heart tube and maybe present in other embryonic tissues. Two Rb RT-PCR

products, A and E, can be amplified from stage 16–18heart tube mRNA but not from stages 3 through 15total embryo mRNA. This observation may arise forseveral reasons. The first is that the lowered annealingtemperature (55°C) used in the total embryo PCR didnot permit specific priming and amplification of vari-ants A and E mRNAs. The second possibility is that theexpression of the A and E isoforms is developmentallyregulated beginning at stage 16 in total chicken em-bryos. The third possibility is that expression of the Aand E isoforms may be both developmentally regulatedand specifically expressed in heart tube at stage 16.Although we cannot resolve these possibilities fromthis RT-PCR analysis, in situ hybridization analysis ofstages 14–20 chicken embryos (see below) indicatesthat Rb mRNA expression in the heart tube increasesdramatically at stage 17–18 providing support for thesecond and third possibilities (see Fig. 4A, panelsC,G,K,O). In summary, our RT-PCR data indicate thatfive Rb mRNA variants are actively expressed in thestage 16–18 embryonic chicken heart, raising the pos-sibility that these variants participate in the morpho-genic processes occurring in this tissue during thistime.

Cloning and Sequence Analysis of Selected RbRT-PCR Products

Based on our Southern blot analysis of the Rb geneand our sequence analysis of the LsRb cDNA showingthat it could arise from alternative splicing of the pub-lished 4.7-kb Rb mRNA, we surmised that the multipleRb RT-PCR products seen in Figure 2 were also alter-natively spliced variants of Rb mRNA. To establishthis, we cloned the Rb RT-PCR products amplified fromheart tube mRNA and subjected them to DNA se-quence analysis. We focused on the largest variant (A)and the two smallest variants (D and E) (C was notanalysed and preliminary sequence analysis of B showsit to be derived from the published 4.7-kb mRNA [Boe-hmelt et al., 1994; Feinstein et al., 1994]). The resultsof our sequence analysis are depicted in Figure 3A,which shows the DNA sequence of the largest RT-PCRproduct, A. Sequence comparison between the Rb RT-PCR products revealed that these mRNA variantsarise from alternative splicing of a higher molecularweight Rb mRNA species, presumably variant A, thelargest variant to be detected by RT-PCR. The splicesites used to generate these variants are numbered inthe sequence with the “exonic” sequences boxed (Fig.3A). All splice sites with the exception of number 7conform to the 59 GU-intron-AG 39 consensus splicejunction dinucleotide sequence (Padgett et al., 1986). Aschematic of the splicing events that give rise to eachvariant is depicted in Figure 3B. Despite our RT-PCRanalysis showing multiple Rb mRNA variants presentin developing chicken embryos, Northern blot analysisof total embryonic mRNA did not provide evidence forthese multiple variants (Fig. 1B). One possible expla-nation for this is that these Rb mRNA variants are of


similar length to and, therefore, indistinguishablefrom, the predominant 4.7-kb Rb mRNA. (Because wehave not determined the complete structure and lengthof the Rb mRNA variants detected in these studies, wecan not presently confirm this.) An alternative andmore likely explanation is that these Rb mRNA vari-ants are expressed at levels too low or in too few cellswithin the embryo to be detected by Northern blotanalysis of total embryo mRNA.

The derived amino acid sequence of each RT-PCRproduct shows that alternative splicing of the 39 end ofthe Rb mRNA generates mRNAs encoding Rb proteinswith different carboxy termini. Computer-based com-positional and structural analyses of these carboxy ter-minal isoforms (DNASTAR Lasergene Navigator pro-grams, DNASTAR, Inc., Madison, WI) did not revealfeatures that would distinguish one isoform from an-other, thereby providing a rationale for alternativesplicing of their mRNAs. However, inspection of theamino acid sequence of each isoform revealed the pres-ence of a recently defined sequence motif, R/KXL(where X is a basic amino acid), that is thought to be a

binding site for cyclin/cyclin-dependent kinase-2(cdk-2) complexes (Adams et al., 1999). The affinity ofcyclin/cdk-2 complexes to bind to and phosphorylate Rbseems to be dependent on the presence of a particularR/KXL motif and/or a critical number of these motifs inthe carboxy terminal of the Rb protein. Interestingly,the alternatively spliced Rb mRNAs described here canencode Rb proteins that differ with respect to the num-ber of KXL cdk binding site motifs in their carboxytermini (Fig. 3B). As a result, these Rb isoforms mayhave different overall binding affinities for cyclin/cdkcomplexes that influence the kinetics of the kinase-Rbinteraction and lead to differential phosphorylationof Rb.

Whole-Mount and Paraffin Section In SituHybridization Studies

Together, Northern blot and RT-PCR analyses of RbmRNA expression in chicken embryos from stages 3through 18 showed that the Rb gene is expressed inearly embryonic tissues, including heart, and that theprimary Rb transcript undergoes alternative splicing

Fig. 2. Reverse transcription polymerase chain reaction (RT-PCR)analysis of Rb mRNA expression in RNA from different stages of chickendevelopment and from stage 16–18 embryonic chicken heart. A: Analy-sis of Rb mRNA expression in total embryo poly A1 RNA from differentstages of development. Blot shows RT-PCR products hybridizing with the1.6-kbp EcoRI Rb coding region probe. B: Analysis of Rb mRNA expres-sion in poly A1 RNA taken from heart tubes isolated from stage 16–18chicken embryos. Numbers at the top of each lane denote the tempera-ture (°C) of the annealing step in each PCR reaction. Blot of RT-PCRproducts was hybridized as in A. Both developmental and heart tissueRT-PCR products were run on the same agarose gel allowing for direct

comparison of RT-PCR band size between heart and total embryo. (Theabsence of the E product in the 60.0°C lane suggests that this RT-PCRproduct did not prime well at annealing temperatures of 60.0°C or below.Because the total embryo PCR reactions were run at an annealingtemperature of 55°C, the E PCR product may not have properly primedleading to its absence.) Reprobing blots with a p107 probe (a tumorsuppressor gene related to Rb) gave no reactivity (data not shown).Some variability in the amounts of the C and D RT-PCR products be-tween developmental stages is evident, but because this was not astrictly quantitative RT-PCR analysis, this finding could be due to differ-ences in RNA template amounts.

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Fig. 4. Whole-mount in situ hybridization of stage 14/15 and stage16–20 chicken embryos with the 1.6 EcoRI Rb coding region riboprobe(antisense and sense). A–C, E–G, I–K, M–O: Embryos hybridized withthe antisense Rb riboprobe. D,H,L,P: Embryos hybridized with the senseRb riboprobe (control embryos). Embryos were photographed at 163magnification (camera-mounted Wild M3Z low-power microscope). Em-

bryos from panels B, F, J, and N were rephotographed at 403 magnifi-cation (panels C, G, K, and O, respectively) to focus on Rb expression inthe heart. B: Isolated hearts from the same embryos shown in A photo-graphed at 403 magnification. Embryos in panels A,D were hybridizedwith sense probe; embryos in panels B–F with antisense Rb probe. Scalebars 5 100 microns in M; 70 microns in O.

Figure 3.

to give multiple mRNA isoforms. To determine whereRb is expressed during development, we performedwhole-mount in situ hybridization studies of stages 10through 20 chicken embryos by using a pan-Rb isoformreactive riboprobe derived from the coding region of theRb cDNA clone originally used to screen the chicken3-day embryonic heart cDNA library (Fig. 4A). PanelsA through C show a stage 14 embryo with little or nospecific hybridization of Rb mRNA when comparedwith the embryo hybridized with the sense Rb controlriboprobe (panel D). When approximately stage 18 orolder embryos were hybridized with antisense Rb ribo-probes, specific hybridization was prominent in theheart tube (panels E–G [stage 17], I–K [stage 18], andM–O [stage 20]) compared with sense riboprobe controlembryos (panels H, L, and P). Upon closer inspection ofthe hearts at higher magnification (panels G, K, and O)and in the same hearts dissected away from the embryoand photographed separately (Fig. 4B, panels B, C, E,and F), it appeared that the most intense hybridizationsignal for Rb mRNA was located in the outflow tract(conus/truncus region) and in the atrioventricular (AV)canal. The expression of Rb mRNA in these regionsappeared to increase with age (compare heart tubesfrom Fig. 4A, panels E, F (approximately stage 17embryo) with panels M, N (approximately stage 20embryo). Although control embryos hybridized with thesense Rb riboprobe (panels H, L, P) showed no stainingin the heart, nonspecific staining was evident in thecentral nervous system. This finding may be due toentrapment of probe within brain vesicles or to theinability to completely wash out probes resulting in lowlevel nonspecific hybridization. In older experimentalembryos (panels J and N), brain staining appears to be

more intense than in control embryos at the samestage, suggesting that Rb is expressed in the earlynervous system, a finding supported by studies show-ing Rb to be involved in regulating the neuronal cellcycle (Lee et al., 1994). The inability to detect RbmRNA in stage 14 embryos, despite its detection byNorthern blot and RT-PCR analyses (see Figs. 1B, 2A),indicates that early stage embryos or regions of laterstage embryos that appear devoid of Rb mRNA hybrid-ization signal may nonetheless express low levels of RbmRNA. This suggests that Rb mRNA is expressed inother tissues of the embryo, but at levels that aresignificantly lower than that seen in the outflow tractand AV canal of the heart.

To resolve in greater detail where Rb mRNA is ex-pressed in chicken embryos, 10-micron-thick paraffintissue sections were prepared from stages 17–18 andstage 20 chicken embryos subjected to in situ hybrid-ization with Rb riboprobes. Inspection of serial sectionsshowed discrete areas of the embryo expressing RbmRNA at high levels: the endocardial cell layer of thelooped heart tube, the mesenchymal cells of endocar-dial cushions within the heart tube, the epithelial layersurrounding the myocardium (epicardium), and themyogenic cells of the somitic myotome (Figs. 5, 6). RbmRNA expression is evident in the endocardial layer(ECL) of the outflow tract (OFT) of a stage 17 embryo(Fig. 5A–C). No Rb mRNA expression is evident in themyocardium (Fig. 5, panel A and higher magnificationimages of panel A not shown). Figure 5E–G shows RbmRNA expression in the ECL of the ventricle of a stage18 embryo. As with the OFT, the ventricular myocar-dium (MYO) is void of Rb mRNA expression (Fig. 5,panel F). Sense Rb riboprobe control experiments forboth stages do not exhibit a hybridization signal (Fig. 5,panels D and H). Rb mRNA is present in cells of theendocardial cushion (ECC) of the atrioventricular(AVC) region of a stage 20 embryo (Fig. 5I–K). Expres-sion seems to be highest in the endocardially derivedmesenchymal cells that have infiltrated the cardiacjelly of the cushion (Fig. 5, panel K [arrows]). Low butdetectable Rb mRNA expression is seen in the epicar-dial cell layer surrounding the myocardium, which it-self appears negative for Rb mRNA (Fig. 5, panel I; seealso Fig. 6B). Figure 5M–O shows Rb mRNA expres-sion in the mesenchymal cells of the endocardial cush-ions of the OFT of a stage 20 embryo. As with theatrioventricular endocardial cushion, Rb mRNA ex-pression appears to be highest in the mesenchymalcells within the cardiac jelly (Fig. 5, panel O [arrows]).Sense Rb riboprobe controls show no hybridizationwith ECC or OFT mesenchymal cells (Fig. 5, panels Land P). The intensity of Rb hybridization signal in theECL of the stage 20 OFT (panel N) appears reducedcompared with that of the ECL in the stage 17 OFT(panel B). This could reflect either reduced Rb expres-sion in ECL cells with development or the exit of Rb-expressing cells out of the ECL due to their transfor-mation to mesenchyme. (Note that the cardiac jelly of

Fig. 3. DNA sequence of reverse transcription polymerase chainreaction (RT-PCR) product A and alternatively spliced isoforms. A: DNAsequence of RT-PCR product A with the alternatively spliced B, D, and Eisoforms shown as derivatives of A (boxed sequences). Underlined se-quences at the 59 and 39 end of the sequence correspond to the se-quence of the primers used in PCR reactions. In-frame stop codons aredenoted in bold. Gray shading indicates the carboxy terminal codingregion of the A Rb isoform. PstI sites used in comparative restriction sitemapping of the LsRb cDNA versus the 4.7-kb Rb mRNA are underlinedat nucleotides 1412 and 2407. (The site at 1412 is missing in the LsRbcDNA relative to the published Rb cDNA [Boehmelt et al., 1994; Feinsteinet al., 1994].) Numbers refer to points where the Rb isoform A mRNA isalternatively spliced. The D RT-PCR product represents the mRNA cor-responding to the LsRb cDNA clone. B: Schematic diagram showing thealternative splicing that gives rise to each Rb mRNA isoform. Filled inboxes represent exonic sequences comprising the 39 end of each RbmRNA isoform; dotted boxes represent exons that may extend furtherdownstream of the 39 primer used in these studies. The location of stopcodons is shown as is the location of the KXL motif described by Adamsand coworkers (1999) to be a cyclin-cdk interaction motif. The location ofthe RT-PCR primers used to amplify these isoforms is depicted by solidarrowheads. Spliced out sequences are denoted by the “V-like” demar-cations extending between exons. Progressive splicing leads to smallerRT-PCR products: Bands A, B, D, and E refer to the RT-PCR products inFigure 2B. The DNA sequence of the Rb RT-PCR product A has beensubmitted to Genbank (Genbank accession no. AF323706)


stage 17 OFT is void of mesenchymal cells, whereasstage 20 OFT contains mesenchymal cells presumablyderived from the ECL.) Together, these data show thatRb mRNA is expressed in the endocardial cell layer ofthe stage 17–18 heart tube and in the mesenchymalcells of the endocardial cushions of the atrioventricularcanal and ventricular outflow tract of the stage 20heart.

Endocardial cushions within the AV canal form thesepta that give rise to the mitral and tricuspid valves ofthe adult heart (Sadler, 1990). In addition to formingthese valves, AV endocardial cushions also participatein the septation of the common atrium into left andright atria by fusing with the septum primum, a tissuecrescent that extends downward from the superoposte-rior wall of the atrium to fuse with endocardial cush-ions. Figure 6A shows sagittal sections of the primitiveatrium of a stage 201 chicken embryo. Expression of

Rb mRNA is apparent in the endocardial layer of theatrium and septum primum (SP) as well as the endo-cardial cell layer of the AV endocardial cushion (ECC)before (Fig. 6A, panels a, b, and d) and after (Fig. 6A,panel c) fusion with the septum primum. Thus, byvirtue of its expression in the endocardial cell layer andmesenchyme of endocardial cushions within the AVcanal, Rb may participate in the formation of the in-teratrial septum as well as in the formation of heartvalves.

Rb mRNA also seems to be expressed in the epicar-dium of the heart and the dermamyotome component ofsomites (Fig. 6, panels B and C). Transverse sections ofa stage 20 heart tube at the level of the atrioventricularcanal show low levels of Rb mRNA expression in cells ofthe epicardium (EPI) (Fig. 6B, panel a; see also Fig. 5,panel I). The epicardium is the epithelial cell layersurrounding the myocardium of the heart. These epi-

Fig. 5. Paraffin sections of chicken embryos hybridized with Rb ribo-probes. A–D: Outflow tract of stage 17 heart. E–H: Ventricle of stage 18heart. I–L: Endocardial cushion of stage 20 heart (atrioventricular canal).M–P: Outflow tract of stage 20 heart. Panels D, H, L, and P are sectionstaken from embryos hybridized with sense Rb riboprobes; all other panelsare sections taken from embryos hybridized with antisense Rb ribo-

probes. OFT, outflow tract; MYO, myocardium; ECL, endocardial celllayer; VENT, ventricle; AVC, atrioventricular canal; ECC, endocardialcushion. Arrows in panel K, L, O, and P are pointing to individual mes-enchymal cells. Scale bars 5 200 microns in A (applies to A,E,I,M), 50microns in B (applies to B,F,J,N), 50 microns in C (applies to C,G,K,O).

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thelial cells can give rise to coronary vascular smoothmuscle cells (CVSMCs) as well as perivascular andintermyocardial fibroblasts. Although the way in whichepicardial cells contribute to the formation of the cor-onary vasculature remains an open question, certainlines of evidence suggest that these cells may firstundergo an epithelial-to-mesenchyme transformation

to form a population of subepicardial mesenchymalcells (SEMCs) situated between the epicardium andmyocardium (Dettman et al., 1998; Perez-Pomares etal., 1997, 1998). The SEMC are then believed to differ-entiate into coronary vascular smooth muscle cells thatcontribute to the formation of the coronary vessels ofthe heart. Molecular markers associated with the epi-

Fig. 6. A: Sagittal sections through stage 20 primitive atrium. Panelsa and b show Rb mRNA expression in the endocardial layer of theseptum primum (SP) and the AV endocardial cushion (ECC). Panel cshows the septum primum fused with the AV endocardial cushion to formthe interatrial septum separating left and right atria. Panel d shows RbmRNA expression in the ECL of the ECC and SP. Scale bars 5 100microns in a, 25 microns in d. B: Transverse sections through theatrioventricular region of a stage 20 heart tube. Panel a: Section from anembryo hybridized with antisense Rb riboprobe showing low level stain-ing in epicardium. Panel B: Section from an embryo hybridized with an Rb

sense control riboprobe. Scale bar: 25 microns in a. C: Transversesection of the trunk of a stage 20 chicken embryo hybridized with anti-sense Rb riboprobe. Panel a: Staining indicating Rb mRNA expressioncan be seen in the somitic myotome (S. MYO) of the neural tube. Panelb is a 403 magnification of the left-hand somitic myotome of panel a.Panel c is a 1003 magnification showing Rb mRNA expression in indi-vidual somitic myotome cells. Scale bars: panel a, 100 microns; panel b,50 microns. SP, septum primum; AT, atrium; EPI, epicardium; S. MYO,somitic myotome; all others, see legend to Figure 5.


thelial-to-mesenchyme transformation of endocardialcells, e.g., JB3 and ES/130, are also expressed on epi-cardial epithelial cells, suggesting that these cells mayundergo an epithelial-to-mesenchyme transformationto form SEMCs in a manner very similar to that ofendocardial cells (Perez-Pomares et al., 1998).

Rb mRNA expression is also evident in migratingsomitic myotome cells (Fig. 6C, panel a, S. MYO). Thedermamyotome is composed of a dorsal layer of der-matome cells that gives rise to mesenchymal connec-tive tissue of the skin and an inner layer of myotomecells that provides myogenic precursors that give riseto skeletal muscles of the back and the limbs. Bothcomponents appear to undergo transformation in thesomite from an epithelial state to a migratory mesen-chymal phenotype during somite dissolution. Althoughthe resolution of our in situ hybridization staining pre-vents us from definitively determining which compo-nent expresses Rb mRNA, we favor myotome cells forthe following reasons. Ordahl and Le Douarin (1992)have shown that the myotome consists of two myogenicpopulations, a resident population that gives rise toaxial muscles and a migratory population that givesrise to muscles of the limbs. Several laboratories haveshown that this latter population expresses the c-metreceptor, a part of the signal transduction pathwaythat we believe may play a role in controlling Rb activ-ity in endocardial cells (see Discussion) (Bladt et al.,1995; Thery et al., 1995; Andermarcher et al., 1996).These observations, taken together with our findingthat Rb mRNA is also expressed in migratory endocar-dial mesenchymal cells, suggest that Rb mRNA is ex-pressed in the migratory muscle precursor cells of themyotome.

DISCUSSION

Our studies show that Rb mRNA is expressed indeveloping chicken embryos as early as stage 3 of de-velopment. We have shown that five different RbmRNA isoforms are expressed throughout chicken em-bryonic development with all isoforms being expressedin the stage 16–18 heart, a period of heart developmentcharacterized by the genetic specification and physicalseptation of the various parts of the heart tube(Mjaatvedt et al., 1999). We demonstrated that theseRb mRNA isoforms arise as a result of alternativesplicing of a primary Rb mRNA transcript. Alternativesplicing of mRNAs is a posttranscriptional mechanismto yield protein isoforms that can differ in activity aswell as tissue distribution (Ferns et al., 1993). The RbmRNA isoforms described in our studies encode Rbproteins having carboxy termini that vary with respectto the number of K/RXL sequence motifs that consti-tute the putative binding sites for cyclin-dependentkinases. Adams and coworkers (1999) have shown thatsequentially reducing the number of these motifs yieldsRb proteins that are progressively less effective as sub-strates for phosphorylation by the cyclin/cdk-2 com-plex. These in vitro observations provide a rationale for

the alternative splicing of Rb mRNA that occurs invivo: alternative splicing of Rb mRNA to give Rb iso-forms with varying numbers of the K/RXL motif (seeFig. 3B) could generate Rb proteins phosphorylated tostoichiometrically different extents and exhibiting ac-tivities that range from fully active (no phosphoryla-tion) to fully inactive (all “phosphoacceptor” sites phos-phorylated). The organization of tissue anlage fromdifferent precursor cell populations may require cells todivide, proliferate, and differentiate at different rates.If differentially phosphorylated forms of Rb can pro-mote differential progression through the cell cycle,then such a mechanism could provide the means forgraded control of the rate of tissue precursor cell pro-liferation. It will be interesting for future studies todetermine whether Rb molecules phosphorylated todifferent extents can differentially regulate a cell’s pro-gression through the cell cycle.

We examined the developmental expression of RbmRNA by Northern blot analysis, RT-PCR, and in situhybridization. Although our Northern blot and RT-PCR data showed Rb mRNA expression as early asstage 3, levels of Rb mRNA detectable by in situ hy-bridization were not attained until approximatelystage 16 of development. By stages 18–20, Rb is highlyexpressed in the outflow tract and AV canal of thedeveloping heart. Analysis of tissue sections takenfrom stage 17–18 and stage 20 embryos hybridized insitu with an Rb coding region riboprobe showed that RbmRNA is expressed in cells of the endocardial layer andmesenchymal cells of the endocardial cushions. Thisexpression pattern is prevalent in two areas of theheart tube in which endocardial cushions form, theatrioventricular canal and the outflow tract (conus/truncus region). The endocardial ridge of the outflowtract cushions fuse to form the aorticopulmonary sep-tum, the first event in a complex process of septationthat separates the aortic from the pulmonary ventric-ular outflow tracts (van den Hoff et al., 1999). As withthe mesenchymal cells populating atrioventricular en-docardial cushions, these cells are also derived fromepithelial cells of the endocardial cell layer that un-dergo an epithelial-to-mesenchyme transformation.The mesenchymal cells expressing Rb in these OFTendocardial cushions are likely to be derived from theOFT endocardium because another source of mesen-chymal cells, the cardiac neural crest, do not enter intothe OFT until stage 21, later than the stages examinedhere (Kirby and Waldo, 1995). In addition, we have notdetected Rb mRNA in migrating neural crest cells.Thus, Rb seems to be involved in the formation ofendocardial cushions in both the ventricular outflowtract and the atrioventricular canal.

Our studies also showed that Rb mRNA is expressedat high levels in the presumptive migratory myogeniccells of the somitic myotome and at lower levels inepicardial cells. Expression of Rb in endocardial andmyotome cells is noteworthy, because these cell typesactively undergo epithelial-to-mesenchyme transfor-

208 WAGNER ET AL.

mation to form populations of migratory and invasivecells that will form heart septa and limb muscle, re-spectively (Eisenberg and Markwald, 1995; Anderma-cher et al., 1996). The presence of Rb in different cellpopulations undergoing epithelial-to-mesenchyme trans-formation suggests that Rb plays a role in this process.Our failure to detect Rb mRNA in other cell typesknown to undergo an epithelial-to-mesenchyme trans-formation, such as neural crest cells, suggests that Rb’srole in epithelial-to-mesenchyme transformation maybe restricted to endocardial, epicardial, and somiticmyotome cells.

Although Rb has been implicated as a transcriptionco-factor in the expression of some genes (Holling-sworth et al., 1993), its most widely accepted functionis that of a cell cycle control molecule. What role, if any,this aspect of Rb function plays in the epithelial-to-mesenchyme transformation of endocardial cells ispresently unclear. One possibility is that Rb may act tosuppress epithelial cell growth to maintain the singlecell endocardial monolayer, while at the same timepromoting mesenchymal cell proliferation. However,three studies have shown that the epithelial cells of theendocardial layer have a proliferative capacity. Thomp-son and colleagues (1995) have shown that both endo-cardial and mesenchymal cells of the atrioventricularcushion express the proliferating cell nuclear antigen(PCNA), a marker for proliferating cells. In studiesusing tritiated thymidine uptake to mark S phase cellsin stage 10–17 chicken embryos, Sissman (1966) notedlabelling of the endocardial layer and attributed thepresence of dividing cells to the necessity of replacingendocardial cells that have migrated out of the layer.More recent thymidine uptake studies carried out inE4 chicken embryos (approximately stage 24) showthat cells in the endocardium as well as the cardiacjelly are actively dividing (Kahane and Kalcheim,1998). Given these findings, one possible role for Rb inthe endocardial cell layer may be to permit sufficientcell division to generate replacement cells in the endo-cardium while at the same time limiting cell division topreclude the expansion of nonactivated or untrans-formed cells.

The high levels of Rb mRNA expression in mesen-chymal cells suggests that Rb may be active in reg-ulating the expansion of the mesenchymal cell pop-ulation to “cellularize” the endocardial cushion.Progression through the cell cycle to promote mesen-chymal cell proliferation and expansion would requireinactivation of Rb by increased phosphorylation. Asdiscussed above, one way to achieve this is by increas-ing the number of cyclin/cdk binding sites in the Rbmolecule to render it a more effective substrate forcyclin-dependent kinases. Another way to regulate Rbphosphorylation is by increasing the levels or activitiesof cyclin/cdk complexes. The intracellular signallingmolecule Ras has been shown to raise cyclin levels inproliferating epithelial and fibroblast cells (Filmus etal., 1994; Winston et al., 1996). Introduction of acti-

vated ras into a rat intestinal epithelium cell line andepithelial cells from mouse mammary gland or 3T3fibroblasts results in dramatic increases in cyclin D1.In the presence of growth factor, cyclin D1 formedcomplexes with cyclin-dependent kinase, leading to in-creased kinase activity and initiation of DNA replica-tion (Winston et al., 1996). These increased cyclin lev-els could lead to the activation of cdks and theconsequent phosphorylation and inactivation of Rb(Peeper et al., 1997). Together, these findings suggestthat growth factor activation of Ras can up-regulatecyclin D1 and cyclin D1/cdk levels, leading to phosphor-ylation of Rb and initiation of DNA synthesis. Althoughsimilar studies have yet to be carried out with mesen-chymal cells, recent studies have implicated a growthfactor receptor capable of activating the Ras signaltransduction pathway in the maintenance and prolif-eration of endocardial mesenchymal cells. The receptorligand, hepatocyte growth factor/scatter factor, orHGF/SF, is a secreted protein that mediates mitogen-esis and cellular motility of epithelial cells and is foundin regions of the developing embryo actively undergo-ing epithelial-to-mesenchyme transformation (Thery etal., 1995; Andermacher et al., 1996). Song and cowork-ers (1997) have shown that HGF/SF is expressed in themyocardial cells underlying the endocardial cushionand that treatment of endocardially-derived cushioncells with HGF/SF results in their increased motilityand proliferation. Binding of HGF/SF to its receptor,the c-met receptor tyrosine kinase, has been shown todirectly activate the MAP kinases linked to activationof the Ras pathway (Ponzetto et al., 1994; Kretzchmaret al., 1997). These findings, together with the demon-strated presence of the c-met receptor on the surface ofendocardially derived mesenchymal cells, suggest thatHGF/SF can potentially activate the Ras signal trans-duction pathway in these cells and, in so doing, stim-ulate their proliferation.

In summary, we have shown that Rb tumor suppres-sor mRNA is expressed throughout early chicken de-velopment with highest expression in the embryonicheart. As a regulator of the cell cycle, Rb may contrib-ute to the formation of tissues by controlling precursorcell proliferation. How Rb achieves this is not known,but the presence of potentially different functional iso-forms of the Rb protein in conjunction with signallingmolecules capable of activating signal transductionpathways that can regulate Rb activity provides evi-dence that dynamic regulation of Rb function is possi-ble. The expression of Rb mRNA in endocardial cellsundergoing an epithelial-to-mesenchyme transforma-tion in the heart suggests that in addition to the phe-notypic conversion of epithelial cells to mesenchyme,expansion of mesenchymal cells to provide sufficientnumbers of cells for formation of the endocardial cush-ions that ultimately form the valves of the heart mayalso be critical to this process. The presence of RbmRNA in different cell types that undergo epithelial-to-mesenchyme transformation attests to a possible


role for the Rb tumor suppressor protein in the expan-sion process.

EXPERIMENTAL PROCEDURES

Low stringency screening of chicken embryo heartcDNA library. A 3-day embryonic chicken heart cDNAlibrary (Stratagene) was screened under low strin-gency conditions by using the 1.6-kbp EcoRI insert of achicken Rb clone as probe (kindly provided by Dr. T.Gilmore, Boston University). A single hybridizingplaque was isolated which preliminary sequence anal-ysis indicated was an Rb cDNA with homology to pub-lished chicken Rb sequences (Boehmelt et al., 1994;Feinstein et al., 1994). This clone was designated LsRb(for “low stringency Rb”).

Genomic DNA Analysis

Newborn chicken liver genomic DNA was digestedwith the appropriate restriction enzymes and 10 mgelectrophoresed in a 0.8% agarose gel for Southern blotanalysis. SalI and XhoI restriction enzymes were in-cluded to bias toward larger restriction fragments be-cause their recognition sequence contains the CG dinu-cleotide that is underrepresented in the genomes ofhigher organisms. Blots were hybridized with the sameprobe used for library screening.

Developmental Northern Blot and TissueSpecificity Northern Blot

Developmental Northern blot. Dissected chickenembryos were staged according to Hamburger andHamilton (1951) and sorted into five groups (stages3–5, 6–8, 9–10, 11–13, and 14–15). Embryos werewashed once, pelleted by centrifugation, and immedi-ately homogenized in RNAzol solution (Tel-Test, Inc.,Friendswood, TX) and RNA isolated according to themanufacturer’s instructions. Equivalent amounts of to-tal RNA (75 mg) were subjected to oligo-dT cellulosechromatography to yield poly A1 RNA. Poly A1 RNAand molecular weight marker RNA (GIBCO-BRL) wereelectrophoresed on a 1% agarose/formaldehyde gel andtransferred to a nylon membrane (Hybond-N). The blotwas hybridized by using as probe a SphI-PstI fragmentof the LsRb clone (corresponding to the Rb pocket do-main and carboxy-terminal end of the coding se-quence).

Tissue specificity Northern blot. Total RNA fromtissues of newborn to 1-day-old chicks was isolated byusing RNAzol (TM). For all tissues, poly A1 RNA wasisolated from 100 mg of total RNA, resolved by gelelectrophoresis and analyzed by Northern blot hybrid-ization under the same conditions as described above.To control for variability in sample loading, blots werereprobed under the same hybridization and wash con-ditions with a cDNA probe encoding the chicken L37aribosomal protein (kindly provided by Dr. T. Tanaka,University of the Ryukyus, Okinawa, Japan). L37a is ahousekeeping gene transcript expressed in all cells andis not developmentally regulated.

RT-PCR

Approximately 150 ng of the poly A1 RNA fromdevelopmentally staged embryos (see above) was usedfor reverse transcriptase-polymerase chain reactions(RT-PCR) (RT-PCR kit of Stratagene). One tenth of thecDNA reaction was used as template in a PCR reaction(cycling parameters: 94°C, 30 sec/55°C, 1 min/72°C, 2min with a 2-sec extension per cycle; 30 cycles). Theprimers used were 59 primer: 59-AAGAGGCTGCGCT-TCGATATAGAA-39, and 39 primer: 59-CAGTCAG-GCGCTAGGAGATCTGTT-39. One-fifth (10 ml) of eachPCR reaction was gel electrophoresed and blotted tonitrocellulose, which was then hybridized with theLsRb clone insert as probe.

For RT-PCR analysis of Rb expression in heart tube,the heart tubes of stage 16–18 chicken embryos werecarefully dissected away from the rest of the embryo soas to ensure no contamination by non-heart tissues.Poly A1 RNA was isolated from heart tubes and con-verted into cDNA in RT-PCR reactions with the sameconditions as described above but with incrementallyhigher annealing temperatures to ensure primer spec-ificity. The PCR reaction products were gel electropho-resed and blotted to nitrocellulose, and the blot wasprobed as described above.

Whole-Mount In Situ Hybridizations

Whole-mount in situ hybridizations were performedon chicken embryos essentially as described (Nieto etal., 1996). Hybridization was usually for 16–18 hr at60°C by using antisense and sense riboprobes derivedfrom the 1.6-kbp EcoRI insert of the chicken Rb cDNA(Feinstein et al., 1994). Embryos were photographed byusing a camera-mounted Wild M3Z low-power micro-scope with Kodak color negative film (Portra 160VC).For sectioning of hybridized embryos, embryos wereembedded in paraffin as described (Nieto et al., 1996)and 10- to 20-micron sections mounted on microscopeslides and photographed by using a Nikon Optiphot-2microscope with Nomarski interference optics. Imageswere captured digitally with the RT Slider digital cam-era system by using SPOT RT software (DiagnosticInstruments, Inc., Sterling Heights, MI).

ACKNOWLEDGMENTS

We thank Dr. T. Gilmore (Boston University) forproviding the chicken Rb coding sequence probe, Dr. T.Tanaka (University of the Ryukyus, Okinawa, Japan)for providing the chicken L37a ribosomal proteinprobe, and Dr. M. Zenke (Max Delbruck Center forMolecular Medicine) for directing our attention to hissubmittal of partial sequence data from Rb mRNAvariants his laboratory found in v-rel transformed bonemarrow cells. We thank Drs. B. Laurent and M.McLeod of the Department of Microbiology and Immu-nology (SUNY) for use of their Nikon microscope anddigital camera and Dorcas Gelabert for preparation ofphotomicrographs. The authors acknowledge Ms.

210 WAGNER ET AL.

Sherry Marmorstein for technical assistance in thesestudies. M.A.Q. S. received support from the N.I.H.

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