Tissue & Cell 35 (2003) 133–142

Relationship between cardiac protein tyrosine phosphorylation andmyofibrillogenesis during axolotl heart development

F. Mengc,1, X.P. Huanga, R.W. Zajdelb, D. Fosterc, N. Dawsonc, S.L. Lemanskia,D. Zawiejac, D.K. Dubeb, L.F. Lemanskia,∗

a Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL 33431, USAb Department of Medicine, Upstate Medical University, Syracuse, NY 13210, USA

c Medical Physiology, Texas A&M University System HSC, College Station, TX 77843, USA

Received 8 August 2002; received in revised form 17 December 2002; accepted 17 December 2002

Abstract

The axolotl,Ambystoma mexicanum, is a useful system for studying embryogenesis and cardiogenesis. To understand the role of proteintyrosine phosphorylation during heart development in normal and cardiac mutant axolotl embryonic hearts, we have investigated the stateof protein tyrosine residues (phosphotyrosine, P-Tyr) and the relationship between P-Tyr and the development of organized sarcomericmyofibrils by using confocal microscopy, two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE)and immunoblotting analyses. Western blot analyses of normal embryonic hearts indicate that several proteins were significantly tyrosinephosphorylated after the initial heartbeat stage (stage 35). Mutant hearts at stages 40–41 showed less tyrosine phosphorylated staining ascompared to the normal group. Two-dimensional gel electrophoresis revealed that most of the proteins from mutant hearts had a lower contentof phosphorylated amino acids. Confocal microscopy of stage 35 normal hearts using phosphotyrosine monoclonal antibodies demonstratedthat P-Tyr staining gradually increased being localized primarily at cell–cell boundaries and cell–extracellular matrix boundaries. In contrast,mutant embryonic hearts showed a marked decrease in the level of P-Tyr staining, especially at sites of cell–cell and cell–matrix junctions. Wealso delivered an anti-phosphotyrosine antibody (PY 20) into normal hearts by using a liposome-mediated delivery method, which resultedin a disruption of the existing cardiac myofibrils and reduced heartbeat rates. Our results suggest that protein tyrosine phosphorylation iscritical during myofibrillogenesis and embryonic heart development in axolotls.© 2003 Elsevier Science Ltd. All rights reserved.

Keywords:Phosphotyrosine; Myofibrils; Confocal microscopy; Cardiogenesis; Axolotl

1. Introduction

During normal embryogenesis, there is a precise controlof events such as cell division, differentiation, and growth.In animals, these events appear to be regulated by proteinphosphorylation through a concerted action of kinases andphosphatases. The phosphorylation of cellular protein ty-rosine residues (P-Tyr) is thought to be important in cellgrowth and differentiation since tyrosine kinase activitiesare expressed by certain growth factor receptors and bythe products of particular proto-oncogenes (for review seeHunter and Cooper, 1985). The roles of the P-Tyr in animalcell embryogenesis have been investigated and the levels of

∗ Corresponding author. Tel.:+1-561-297-0475; fax:+1-561-297-0422.E-mail address:lemanski@fau.edu (L.F. Lemanski).1 Present address: Department of Molecular and Cell Biology, University

of Texas at Dallas, Richardson, TX, USA.

protein Tyr phosphorylation have been shown to accompanyembryo development in vertebrates (Maher and Pasquale,1988; Turner, 1991, 1994; Nieto et al., 1992; Snider, 1994)and invertebrates (Shilo, 1992; Perrimon, 1993).

The axolotl,Ambystoma mexicanum, is an intriguing ani-mal model because it offers several advantages for studyingembryogenesis and cardiogenesis. Although the axolotl is acold-blooded vertebrate, it appears to undergo, at the cell andtissue levels, the same developmental processes as highervertebrates, including mammals (Lemanski et al., 1997). Inaddition, a naturally occurring recessive mutation inA. mex-icanumresults in an absence of normal heart contractions inaffected embryos (Humphrey, 1972); the gene that causesthis mutation was designatedc for “cardiac lethal.” At stage34, about 1 week after fertilization of the eggs, the normalhearts first start to contract. This is the earliest stage at whichthe mutant embryos (c/c) with non-beating hearts can bedistinguished from their normal siblings (+/+ or +/c) in a

0040-8166/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0040-8166(03)00012-0

134 F. Meng et al. / Tissue & Cell 35 (2003) 133–142

heterozygous spawning (+/c × +/c). The mutant embryosdevelop for approximately 3 weeks after the heartbeat stagebut eventually die from a lack of circulation (stages 41–42).Previously, we reported cardiomyofibrillar protein changesduring cardiogenesis in axolotl (Lemanski et al., 1996,2001). In the present study, we have investigated the tempo-ral and spatial patterns of phosphotyrosine immunolocaliza-tion during cardiac myofibrillogenesis in axolotl hearts. Theresults demonstrate that the P-Tyr levels increase signifi-cantly in axolotl hearts during embryonic development. Mu-tant axolotl hearts show less P-Tyr as compared to the normalones. We also introduced an anti-phosphotyrosine antibody(PY 20) into normal hearts, which disrupted the existingcardiac myofibrils and resulted in reduced heartbeat rates.Introduction of a C-protein monoclonal antibody (ALD 66)did not result in a disruption of organized myofibrils. Thesedata suggest that tyrosine phosphorylation plays an impor-tant role in myofibrillogenesis during heart developmentin axolotls.

2. Materials and methods

2.1. Procurement and maintenance of axolotls

Normal and cardiac mutant axolotl embryos were ob-tained from matings between homozygous normal (+/+ ×

+/+) or heterozygous (+/c × +/c) animals from ourcolony at the TAMU Health Science Center (College Sta-tion, TX). The animals were maintained in aquaria in di-luted Holtfreter’s solution (29 mM NaCl, 0.45 mM CaCl2,0.33 mM KCl, 0.1 mM MgSO4, and 4.76 mM NaHCO3),fed commercial salmon pellets and supplemented occa-sionally with raw beef liver and live earthworms. The em-bryos were staged according to the standard staging system(Bordzilovskaya et al., 1989).

2.2. Western blot analysis

Normal embryos (stages 32–43) and mutant embryos(stages 40–41) were washed four times in filter-sterilizedSteinberg’s buffered salt solution (58 mM NaCl, 0.67 mMKCl, 0.9 mM CaCl2, 0.2 mM MgSO4, 4.6 mM HEPES, pH7.4) containing 1% antibiotic/antimycotic at a final con-centration of penicillin G sodium 100 U/ml, streptomycinsulfate 0.1 g/ml, and amphotericin B 0.25�g/ml (Gibco LifeTechnologies, Grand Island, NY). The heart cavities of thenormal and mutant embryos were opened up by removingthe skin ventral to the hearts. After extirpation, the heartswere homogenized and lysed by incubation for 20 min in1 ml of ice-cold lysis buffer containing 20 mM Tris–HCl(pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM phenyl-methylsulfonyl fluoride, 0.15 U/ml aprotinin, 10�g/ml leu-peptin, 10�g/ml pepstain A, 1 mM sodium orthovanadate,and 10% glycerol. The lysate was clarified by centrifugationat 14,000×g for 10 min at 4◦C. For immunoblotting, equal

amounts of protein (50�g) from whole-heart lysates wereboiled for 5 min in a SDS sample buffer (Novex, San Diego,CA). Proteins were fractionated by SDS-polyacrylamide gelelectrophoresis (PAGE) on precast 4–12% gradient minigelsand transferred to nitrocellulose sheets (blots). Blots werestained with 0.1% Ponceau S solution to visualize proteinbands and confirm both consistent protein loading amongwells and complete transfer of proteins to blots. The blotswere then blocked by incubation in 10 mM Tris–HCl (pH7.5), 100 mM NaCl, 0.1% Tween-20 and 1% bovine serumalbumin (BSA) overnight at 4◦C. To examine tyrosine phos-phorylation, the samples were further incubated with PY20:HRPO anti-phosphotyrosine antibody conjugated withhorseradish peroxidase (Transduction Laboratories, Lex-ington, KY) for 2 h at room temperature and washed threetimes. Immunoreactive bands were detected by enhancedchemiluminescence reagents (Amersham Life Science, IL)and exposed to Kodak X-ray film for 15 s.

In each experiment, samples were sized by comparisonto standard molecular weight markers run in parallel. Theconcentration of protein in cell lysates was determined withBradford’s method using the Bio-Rad protein assay reagentwith BSA as a protein standard. Two control studies wereperformed to show the specificity of antibody binding:(1) immunoblots were incubated with PY 20:HRPO togetherwith 20 mM O-phospho-l-tyrosine (Sigma), a competitiveinhibitor of phosphotyrosine binding; and (2) blots wereincubated with purified normal IgG control. Each Westernblot analysis was repeated at least three times.

2.3. Two-dimensional SDS-PAGE

Two-dimensional gel analysis of tyrosine phosphorylatedproteins was performed using the Bio-Rad mini-PROTEANsystem according to the manufacturer’s instructions. Sam-ples for two-dimensional gel analysis were prepared as de-scribed for Western blot analysis, except in this case theextracts were diluted 1:1 in two-dimensional sample buffer(9.5 M urea, 2.0% Triton X-100, 5%�-mercaptoethanol,1.6% Biolyte 3/10 and 0.4% Biolyte 8/10) prior to isoelectricfocusing (IEF). Axolotl embryonic proteins were separatedby IEF over a pH range of 3–10 with enhanced resolutionin the basic region due to a 1:5 ratio of the ampholytes, Bi-olyte 8/10 and 3/10 (Bio-Rad) in tube gels. After IEF, thetube gels were overlayed onto a SDS-12% polyacrylamidegel and the axolotl proteins again separated by SDS-PAGE.Following PAGE, the proteins were transferred to nitrocel-lulose and tyrosine-phosphorylated proteins were detectedas described before.

2.4. Normal whole-heart cultures

Normal embryos from stages 36–38 were washed fourtimes in filter-sterilized Steinberg’s solution. Embryos werekilled by cervical dislocation, and the hearts were extirpatedwith watchmaker’s forceps using a dissecting microscope

F. Meng et al. / Tissue & Cell 35 (2003) 133–142 135

and initially placed into Steinberg’s solution until treatment.The culture medium was Steinberg’s solution containing 1%antibiotic/antimycotic (Gibco: penicillin G sodium 1000 U,1 mg streptomycin sulfate, and 2.5�g amphotericin B perliter). Organ cultures were maintained in a humid chamberat 17◦C for the specified time. Whole-heart cultures wereexamined daily for the presence of beating.

2.5. Liposome-mediated delivery of PY 20phosphotyrosine antibody

Transfections were performed using lipofectin (Life Tech-nologies) transfection reagent according to manufacturer’sprotocols. An anti-phosphotyrosine monoclonal antibody(PY 20) was delivered into normal hearts. Briefly, 15�l oflipofectin 1 mg/ml was added to 35�l of Steinberg’s solu-tion for 30 min at room temperature. Fifteen microliters ofPY 20 antibody was added to 35�l of Steinberg’s solutionand mixed with the lipofectin solution for 15 min (PY 20 fi-nal concentration of 0.0087�g/�l). The hearts reacted withPY 20 were then placed into 10�l drops. Control heartswere cultured and placed through the same steps as the PY20 hearts but without the antibody. Other control hearts weresimply cultured for 4 days in Steinberg’s solution alone.Beating was monitored in the normal hearts over the entireculture period. C-protein (ALD 66) antibody (Sigma) wasalso transfected into normal hearts as a positive control toconfirm that an antibody by itself does not disrupt myofibrilformation and beating. This protein has not been found tobe expressed in embryonic axolotl hearts at this stage.

2.6. Confocal microscopy

The cardiac tissues were fixed according to a previouslydescribed method (Bell et al., 1987). All steps took placeat room temperature with gentle agitation on an orbitalshaker. Hearts were placed into 1 mM dithiosulfylpropi-onate (Sigma) dissolved with 1% dimethylsulfoxide inSteinberg’s solution for 15 min. Further permeabilizationoccurred in 0.5% Nonidet P-40 in Steinberg’s solutionfor 15 min. The reaction was quenched by two 10-minwashes in 0.1 M glycine. After blocking with 3% BSA inSteinberg’s solution containing 0.05% Tween-20 for 1 h, thehearts were incubated in 1:75 PY 20 purified monoclonalanti-phosphotyrosine antibody (Transduction Laboratories)or purified monoclonal anti-tropomyosin antibody (Linet al., 1985) diluted to 1:75 in Steinberg’s solution for 1 h,and washed several times. Hearts were then incubated inOregon green-conjugated goat anti-mouse secondary anti-body (Molecular Probes, Eugene, OR) at a 1:75 dilution for45 min. The hearts were rinsed in several changes of BSA inSteinberg’s solution and then postfixed in 2% paraformalde-hyde for 30 min. The fixation was quenched by 0.1 M glycinefor 30 min and then placed in Steinberg’s solution. Thehearts were mounted on slides in 50% glycerol/phosphatebuffered saline containing 2%n-propyl gallate. Three layers

of fingernail polish were used to support the glass cover-slips to prevent crushing of the whole hearts. The specimenswere viewed on a Meridian Instruments Ultima Z ConfocalLaser Scanning Microscope. Fluorescence was excited at530 nm. A 30 section Z-series was made for each sampleand system software was used to display the final imageon a monitor. Color photographs of these images were thenprinted on Ilfochrome Classic Deluxe CLM 1k paper.

3. Results

3.1. Temporal changes of P-Tyr in the heart duringembryonic development

Western blot analyses have shown an overall increaseof tyrosine-phosphorylated proteins in normal hearts fromstages 32–43 (Fig. 1). The major hyperphosphorylated bandshad molecular masses of 150–160, 110–120, 85–90, 60–70,and 45–50 kDa. However, the phosphotyrosine staining wassignificantly decreased in mutant axolotl hearts at stages38–39 compared to the normal hearts at the same stages(Fig. 1). To further visualize changes in the tyrosine phos-phorylation pattern with heart development, we analyzedthe proteins obtained from stages 42–43 (Fig. 2a) and 32–33(Fig. 2b) whole-heart extracts on two-dimensional gels, fol-lowed by Western blotting and staining with PY 20:HRPOanti-phosphotyrosine antibody conjugated with horseradishperoxidase. Compared with the pre-heartbeat stage, thepost-hatching stage hearts showed an overall increase oftyrosine phosphorylated proteins, whose molecular weightsranged between 40 and 120 kDa, with predominately acidicisoforms. The results suggest that hearts at the post-hatchingdevelopmental stage contain a higher content of phospho-rylated amino acids than the pre-heartbeat organs.

3.2. Spatial distribution of P-Tyr proteins in the heartduring embryonic development

Fig. 3 shows stereoconfocal images of optical Z-seriescollected from whole mounted hearts stained with PY 20anti-phosphotyrosine antibody from the pre-heartbeat stagethrough post-hatching developmental stages. At stages32–33, the hearts exhibited slight phosphotyrosine stainingin the form of small amorphous collections in some, butnot in all areas. Ventricular regions showed a weaker P-Tyrsignal than in other areas (Fig. 3a). From initial heart-beat stages 35–36 (Fig. 3b) to post-hatching stages 42–43(Fig. 3d), P-Tyr staining intensity was gradually increased.A stronger P-Tyr staining was observed in the hearts atpost-hatching stages (Fig. 3d) than that at pre-heartbeatstages (Fig. 3a). The enhanced tyrosine phosphorylationsites were primarily localized at cell–cell boundaries andcell–extracellular matrix boundaries. The competitive P-Tyrantibody blocker,O-phospho-l-tyrosine prevented almostall the antibody binding (Fig. 3e). Since mutant axolotl

136 F. Meng et al. / Tissue & Cell 35 (2003) 133–142

Fig. 1. Temporal increase of P-Tyr in the heart during embryonic development. Whole-heart homogenates from normal embryos (stages 32–43) areanalyzed by Western blotting with PY 20:HRPO anti-phosphotyrosine antibody conjugated with horseradish peroxidase. The tyrosine phosphorylationstaining is gradually increased after heartbeat stages (stages 35–36). The phosphotyrosine staining is significantly decreased in mutant axolotl hearts atstages 38–39 compared to the normal hearts at the same stages. Positions of molecular mass markers are indicated on the left. Results shown representthree independent experiments.

hearts contain much less organized myofibril structures,only a few spots were observed at the surfaces of the cardiacmutant cells at stages 35–36 (Fig. 3f) and 38–39 (Fig. 3g).The staining in mutant hearts was significantly lower thanthat in normal hearts of the same stages (Fig. 3b and c).

At a higher magnificent (1240×), P-Tyr staining wasfound mainly at or near the cell peripheries of the ax-olotl hearts at stages 32–33 (Fig. 4a). The cell peripheriesstained intensely at stages 35–36, the initial heartbeatstages (Fig. 4b). Thick bundles and immature myofibrilswith periodic “dots” appeared. Together with thick bun-dles, “particulate” P-Tyr expression was serially alignedin the circumferential direction of the heart at this stage(Fig. 4b). At stages 38–39, along the “belt-like” sites ofP-Tyr localization and other cell–cell boundaries of bothlayers, “clumped-type” P-Tyr expression became more def-inite (Fig. 4c). Mature interlaced myofibril-like structures

Table 1Inhibition of the heart-beating rate and sarcomeric myofibril formation in normal hearts by introduction of PY 20 phosphotyrosine antibody into cardiaccells

Treatment Heartbeat rate (beats/min) Hearts with organized myofibrilstructure/total tested hearts

PY 20 group (n = 9) 7.1 ± 0.8∗,∗∗ 1/9∗,∗∗

Control, C-protein group (n = 6) 24.7± 2.3 6/6Control, lipofectin group (n = 4) 25.3± 2.1 4/4Control, Steinberg’s group (n = 4) 25.6± 1.9 4/4

∗ P < 0.01 compared to Steinberg’s controls.∗∗ P < 0.01 compared to lipofectin controls.

constituted dense networks at post-hatching stages 42–43(Fig. 4d). However, P-Tyr is nearly undetectable in theventricle region of adult axolotl heart (Fig. 4e).

3.3. Phosphotyrosine antibody (PY 20) inhibitsmyofibrillogenesis and heart-beating rate

To further confirm the role of tyrosine phosphorylationin heart development, we introduced PY 20 monoclonalantibody into normal hearts at stage 36. After a 4-day in-cubation, we found that heart-beating rate was significantlydecreased in PY 20 treated hearts (Table 1). Moreover, weobserved under confocal microscopy the transfected heartsas compared to the controls either treated with C-proteinantibody or only incubated with lipofectin solution alone(Fig. 5). Normal hearts showed a clear striated cardiacmuscle pattern and myofibril structure after staining with

F. Meng et al. / Tissue & Cell 35 (2003) 133–142 137

Fig. 2. Phosphotyrosine protein profiling in normal axolotl hearts. Proteins from whole-heart extracts were separated according to their isoelectric points(one-dimensional, pH 3–10) and molecular weights (two-dimensional). After separation in the two-dimension, the proteins were blotted onto nitrocelluloseand stained with PY 20:HRPO anti-phosphotyrosine antibody conjugated with horseradish peroxidase. (a) The P-Tyr containing protein profile in axolotlhearts at post-hatching stages (stages 42–43) and (b) the P-Tyr containing protein profile in axolotl hearts at pre-heartbeat stages (stages 32–33). Theproteins obtained from all samples were carefully aligned according to their migration during IEF.

anti-tropomyosin antibodies (Fig. 5a). After treatment withPY 20 antibodies, the hearts showed a disruption of sarcom-eric structure (Fig. 5c). Moreover, the PY 20 treated normalhearts had positive staining for PY 20 in amorphous col-lections and on linear sarcomeric structures, indicating thatPY 20 antibodies were well introduced into cardiac cells(Fig. 5b), whereas the hearts transfected with C-proteinantibody or lipofection in Steinberg’s did not have positivePY 20 staining (results not shown). Normal hearts main-tained in Steinberg’s alone did not have a decrease in thebeating rate after 4-day incubation and these control heartswere positive for tropomyosin staining (results not shown).Liposome-mediated delivery of C-protein antibody intonormal hearts did not result in a decrease in heart-beatingrates after 48- or 72-h incubation. ALD 66/lipofectin-treatedhearts had a normal heart-beating rate as compared to thatof controls (Table 1). The penetration of ALD 66 antibodywas confirmed by incubation of these hearts with a sec-ondary antibody, which showed positive staining around theventricular area (Fig. 5d). The well-organized sarcomeric

myofibrils in ALD 66/lipofectin-treated hearts decoratedby anti-tropomyosin antibodies were observed throughoutthe entire heart (Fig. 5e). These data suggest that intro-duced PY 20 antibodies specifically prevent the formationof sarcomeric structure in the developing hearts.

4. Discussion

The axolotl provides a valuable model system for studyingheart development and function. The eggs acquired from aspawning are large enough for surgical manipulation and theheart primordia is readily accessible. Most importantly, theaxolotl also carries a cardiac-lethal mutation (genec), whichresults in the absence of myofibril assembly in the heart, butnot in skeletal muscle. The myocardium of the mutants un-dergoes abnormal sarcomere development and the hearts failto beat. In the present study, we have investigated temporaland spatial patterns of phophorylated tyrosine immunolo-calization during initial myofibrillogenesis of the Mexican

138 F. Meng et al. / Tissue & Cell 35 (2003) 133–142

Fig. 3. Spatial distribution of P-Tyr proteins in the heart. Three-dimensional confocal micrographs (best viewed with the aid of red-green glasses) ofwhole-mount preparations of normal hearts, stained with PY 20 anti-phosphotyrosine antibody, at different developmental stages: 32–33 (a), 35–36 (b),38–39 (c), 42–43 (d), as well as mutant hearts (c/c) at stages 35–36 (f) and stages 38–39 (g). Normal hearts, incubated with PY 20 antibody plus acompetitive phosphotyrosine blockerO-phospho-l-tyrosine, are shown as a negative control (e); 1240×.

F. Meng et al. / Tissue & Cell 35 (2003) 133–142 139

Fig. 4. Spatial distribution of P-Tyr proteins in cardiac cells. Confocal three-dimensional observation of the heart ventricular areas (best viewed with theaid of red-green glasses), which have been stained with PY 20 antibody, from normal axolotl hearts at different stages: 32–33 (a), 35–36 (b), 38–39(c), 42–43 (d) and adult axolotl hearts (e). Dense networks composed of interlaced myofibrils have been gradually established from pre-heartbeat stage32–33 (a) to post-hatching stage 42–43 (d). Much less P-Tyr staining is detected in the ventricle region of adult axolotl heart (e). These are stereoscopicimages consisting of 30 serial optical sections of inner layer myocardial cells; 1240×.

axolotl embryonic heart. We have also detected the pro-teins containing phosphorylated tyrosine by immunoblot-ting on one- and two-dimensional separated protein samplesfrom axolotl hearts at different developmental stages. Theresults demonstrate that the P-Tyr levels increase signifi-cantly in axolotl embryonic hearts from the pre-heartbeat topost-hatching stages and then disappear in the adult axolotlhearts. Mutant axolotl hearts show much less P-Tyr com-pared to that of the normal hearts at stages 35–36, 38–39and 40–41. The results from one- and two-dimensional im-munoblotting experiments show that the proteins containingP-Tyr are in the molecular range of 45–120 kDa and local-ized at the acidic phase. These data suggest that tyrosine

phosphorylation plays an important role during myofibrillo-genesis in embryonic axolotl hearts.

In normal axolotl hearts, the concentration of P-Tyr ishigh during the early stages of embryonic development.During late embryonic development, the P-Tyr concentra-tion decreases and finally falls to very low levels in adulthearts. These results are consistent with other reports onchick embryonic tissues (Maher and Pasquale, 1988; Takataand Singer, 1988). Patstone and Maher reported that P-Tyrwas detected in a population of cells that were beginning todifferentiate rather than in undifferentiated cells (Patstoneand Maher, 1993). These results suggest that P-Tyr is relatedto cell growth and differentiation. In normal axolotl hearts,

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Fig. 5. Anti-phosphotyrosine antibody (PY 20) inhibits myofibrillogenesis in axolotl hearts. (a) Normal hearts with anti-tropomyosin staining showswell-organized sarcomeric myofibrils throughout the whole heart. (b) Normal hearts have been transfected with PY 20 phosphotyrosine antibodyand lipofectin. The penetration of the antibody is confirmed by incubating these hearts with a secondary antibody, which shows positive stainingaround the ventricular area in amorphous collections. (c) In normal heart treated with lipofectin and PY 20 phosphotyrosine antibody, well-formedmyofibrils are disrupted and tropomyosin levels are decreased as detected with an anti-tropomyosin antibody. (d) Normal hearts have been trans-fected with ALD 66 antibody and lipofectin. The penetration of the antibody is confirmed by incubating these hearts with a secondary antibody,which shows positive staining around the ventricular areas. (e) In normal hearts treated with ALD 66 antibody and lipofectin, well-organized sar-comeric myofibrils can be seen throughout the cells of the hearts stained by conventional immunofluorescence with anti-tropomyosin antibody;6480×.

we observed the localization of P-Tyr staining comparedwith the arrangement of myofibrils in the heart as shownby antibody staining with anti-tropomyosin. The P-Tyrstaining pattern is very similar to the tropomyosin stainingpattern. As shown inFig. 4 of our study, the P-Tyr labelingis observed clearly near the myocardial outer surface, which

is also very similar to F-actin and vinculin labeled earlychick embryonic hearts in Tokuyasu’s pioneer studies oncardiac myofibrillogenesis (Tokuyasu, 1989). We previouslyreported that mutant axolotl hearts lack organized myofib-rils and show little tropomyosin staining (LaFrance andLemanski, 1994). In the present study, we found that P-Tyr

F. Meng et al. / Tissue & Cell 35 (2003) 133–142 141

concentration in mutant hearts was much less than normalafter the initial heartbeat stage. All these data suggest thatthe proteins containing P-Tyr might be myofibril proteinsor the proteins which regulate myofibrillogenesis duringheart development. Liposome-mediated delivery of ananti-phosphotyrosine antibody bound to the phosphotyro-sine protein in cardiac cells and disrupted the contractionsin normal hearts. These results confirm the results ofZajdelet al. (1998)that a protein can be introduced by cationicliposomes into the cardiomyocytes and intact embryonicaxolotl hearts. Myofibril formation was disrupted in thenormal hearts after 4-day incubation with the PY 20 anti-bodies. The heart-beating rates were also decreased in thetreated hearts. Introduction of a C-protein antibody that isnot normally expressed in embryonic axolotl hearts at thisstage did not result in a disruption of beating or myofibrilformation. Therefore, an antibody that is nonspecific foraxolotl cardiac proteins did not cause the same results as weobserved on phosphotyrosine inhibited by PY 20 antibodies.These results suggest that tyrosine phosphorylation plays acritical role in myofibrillogenesis in developing hearts.

Protein tyrosine phosphorylation is a feature common tomany signal transduction pathways that mediate cell pro-liferation and development. The intensity and duration oftyrosine phosphorylation during heart development is deter-mined by the relative activities of protein tyrosine kinasesand protein tyrosine phosphatases. It has been demonstratedthat expression of a dominant negative mutant of fibroblastgrowth factor receptor-1 (FGFR-1) in Xenopus embryosresults in disruption of mesoderm formation and posteriordevelopment (Amaya et al., 1991). In addition, targeted dis-ruption of the genes of many members of the ‘src’ family oftyrosine kinases also results in severe abnormalities of earlymouse development (Soriano et al., 1991; Stein et al., 1994).Recently, gp130-dependent signaling pathways have beenimplicated in the heart development and the progression ofcardiac hypertrophy (Yoshida et al., 1996; Kuwahara et al.,1999). gp130 is a signal transducting receptor componentshared by interleukin 6, interleukin 11, leukemia inhibitorfactor, and cardiotrophin 1. The activation of the JAK fam-ily of cytoplasmic tyrosine kinases in gp130-dependentsignaling pathways leads to subsequent tyrosine phospho-rylation and functional activation of a latent transcriptionfactor, APRF/STAT3 and the Ras/mitogen-activated proteinkinase (MAPK) cascade (Yoshida et al., 1996). Furtherstudies will be required to determine the precise role andmechanism of action of tyrosine kinase activity in axolotlhearts at different developmental stages and to identify whatrole tyrosine kinase has in the process of normal cardiacmyofibrillogenesis.

Acknowledgements

Supported by NIH Grants HL-58435 and HL-061246 toLarry Lemanski.

References

Amaya, E., Musci, T.J., Kirschner, M.W., 1991. Expression of a dominantnegative mutant of the FGF receptor disrupts mesoderm formation inXenopusembryos. Cell 66, 257–270.

Bell, P.B., Rundquist, I., Svensson, I., Collins, V.P., 1987. Use of cytoflu-orotomy to evaluate binding of antibodies to the cytoskeleton of cul-tured cells. J. Histochem. Cytochem. 35, 1381–1388.

Bordzilovskaya, N.P., Detlaff, T.A., Duhon, S.T., Malacinski, G.M., 1989.Developmental-stage series of axolotl embryos. In: Armstrong, J.B.,Malacinski, G.M. (Eds.), Developmental Biology of the Axolotl. Ox-ford Press, New York, pp. 201–219.

Humphrey, R.R., 1972. Genetic and experimental studies on a mutant gene(c) determining absence of heart action in embryos of the Mexicanaxolotl (Ambystoma mexicanum). Dev. Biol. 27, 365–375.

Hunter, T., Cooper, J.A., 1985. Protein tyrosine kinases. Annu. Rev. Biol.Chem. 54, 897–930.

Kuwahara, K., Saito, Y., Harada, M., Ishikawa, M., Ogawa, E., Miyamoto,Y., Hamanaka, I., Kamitani, S., Kajiyama, N., Takahashi, N., Naka-gawa, O., Masuda, I., Nakao, K., 1999. Involvement of cardiotrophin-1in cardiac myocyte-nonmyocyte interactions during hypertrophy of ratcardiac myocytes in vitro. Circulation 100, 1116–1124.

LaFrance, S., Lemanski, L.F., 1994. Immunofluorescent confocal analysisof tropomyosin in developing hearts of normal and cardiac mutantaxolotls,Ambystoma mexicanum. Int. J. Dev. Biol. 38, 695–700.

Lemanski, L.F., Meng, F., Lemanski, S.L., Dawson, N., Zhang, Z., Foster,D., Li, Q., Nakatsugawa, M., Zajdel, R.W., Dube, D.K., Huang, X.P.,2001. Creation of chimeric mutant axolotl: a model to study earlyembryonic heart development in Mexican axolotls. Anat. Embryol.203, 335–342.

Lemanski, L.F., Nakatsugawa, M., Bhatia, R., Erginel-Unaltuna, N., Spin-ner, B.J., Dube, D.K., 1996. A specific synthetic RNA promotes car-diac myofibrillogenesis in the Mexican axolotl. Biochem. Biophys.Res. Commun. 229, 974–981.

Lemanski, L.F., Zajdel, R.W., Nakatsugawa, M., Bhatia, R., Spinner, B.J.,Fransen, M.E., Gaur, A.F., McLean, M.D., Lemanski, S.L., Dube, D.K.,1997. Molecular biology of heart development in the Mexican axolotl,Ambystoma mexicanum. J. Tsitol. (Cytology) 39, 918–927.

Lin, J.J.C., Chou, C.S., Lin, J.L.C., 1985. Monoclonal antibodies againstchicken tropomyosin isoforms: production, characterization, and appli-cation. Hybridoma 4, 223–242.

Maher, P.A., Pasquale, E., 1988. Tyrosine phosphorylated proteins indifferent tissues during chick embryo development. J. Cell Biol. 106,1747–1755.

Nieto, M.A., Gilardi-Herbenstreit, P., Charnay, P., Wilkinson, D.G., 1992.A receptor protein tyrosine kinase implicated in the segmental pattern-ing of the hindbrain and mesoderm. Development 116, 1137–1150.

Patstone, G., Maher, P.A., 1993. Phosphotyrosine-containing proteins areconcentrated in differentiating cells during chicken embryonic devel-opment. Growth Factors 9, 243–252.

Perrimon, N., 1993. The torso receptor protein-tyrosine kinase signallingpathway: an endless story. Cell 74, 219–222.

Shilo, B.Z., 1992. Roles of receptor tyrosine kinases inDrosophiliadevelopment. FASEB J. 6, 2915–2922.

Snider, W.D., 1994. Functions of the neurotropins during nervous sys-tem development: what the knockout are teaching us. Cell 77, 627–638.

Soriano, P., Montgomery, C., Geske, R., Bradeley, A., 1991. Targeteddisruption of thec-src proto-oncogenes leads to osteopetrosis in mice.Cell 64, 693–702.

Stein, P.L., Vogel, H., Soriano, P., 1994. Combined deficiencies f src,fyn, and yes tyrosine kinases in mutant mice. Genes Dev. 8, 1999–2007.

Takata, K., Singer, S.J., 1988. Phophotyrosine-modified proteins are con-centrated at the membranes of epithelial and endothelial cells duringtissue development in chick embryos. J. Cell Biol. 106, 1757–1764.

142 F. Meng et al. / Tissue & Cell 35 (2003) 133–142

Tokuyasu, K., 1989. Immunocytochemical studies of cardiac myofibril-logenesis in early chick embryos. III. Generation of fasciae adherentsand costameres. J. Cell Biol. 108, 43–53.

Turner, C.E., 1991. Paxillin is a major phosphotyrosine-containing proteinduring embryonic development. J. Cell Biol. 115, 201–207.

Zajdel, R.W., McLean, M.D., Lemanski, S.L., Muthuchamy, M., Wiec-zorek, D.F., Lemanski, L.F., Dube, D.K., 1998. Ectopic expressionof tropomyosin promotes myofibrillogenesis in mutant axolotl hearts.Dev. Dyn. 213, 412–420.

Yoshida, K., Taga, T., Saito, M., Suematsu, S., Kumanogoh, A., Tanaka,T., Fujiwara, H., Hirata, M., Yamagami, T., Nakahata, T., Hirabayashi,T., Yoneda, Y., Tanaka, K., Wang, W., Mori, C., Shiota, K., Yoshida,N., Kishimoto, T., 1996. Targeted disruption of gp130, a commonsignal transducer for the interleukin 6 family of cytokines, leads tomyocardial and hematological disorders. Proc. Natl. Acad. Sci. U.S.A.93, 407–411.