Desmin and titin expression in early postimplantation ... · Titin was found in the early heart anlage at stage 8.25 d.p.c, when no desmin expression was observed in this tissue

Development 107, 585-596 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

585

Desmin and titin expression in early postimplantation mouse embryos

G. SCHAART1*, C. VIEBAHN2, W. LANGMANN2 and F. RAMAEKERS1

^Department of Pathology, University Hospital Nijmegen, Geert Crooteplein Zuid 24, 6525 GA Nijmegen, The Netherlands2Institute of Anatomy, University of Bonn, Nussallee 10, D-5300 Bonn, Federal Republic of Germany

* Author for correspondence

Summary

The expression of the intermediate filament (IF) con-stituents desmin, vimentin and keratin, as well as thestriated-muscle-specific marker titin, was studied inmouse embryos of 8.0 to 9.5 days post coitum (d.p.c),using the indirect immunofluorescence technique incombination with polyclonal and monoclonal antibodies.During the development of the embryo, desmin was firstdetected at 8.25 d.p.c. in the ectoderm, where it wastransiently coexpressed with keratin and vimentin. Atlater stages, the ectoderm contained only keratin and toa certain extent also vimentin IF. At 8.5 d.p.c, desminwas found exclusively in the heart rudiment, andremained present with increasing intensity in the myo-cardial cells during later cardiogenesis. Striation ofdesmin in the heart muscle cells was observed in 9.5d.p.c. embryos. At these stages (8.5-9.5 d.p.c), tripleexpression of the IF proteins desmin, vimentin andkeratin was evident in these cells. From 9.0 d.p.c.onwards, desmin could be detected in the myotomes aswell. Immunoblotting studies of 9.5 d.p.c. mouse em-bryos confirmed the immunohistochemical data.

Titin was found in the early heart anlage at stage 8.25d.p.c, when no desmin expression was observed in thistissue. At this stage the titin appeared in a punctatepattern, similar to that observed in cardiac myofibrils ofearly chicken embryos (Tokuyasu and Maher, 1987; J.CeUBiol. 105, 2781-2793). In 8.5 d.p.c. mouse embryos,this punctate titin staining pattern was still observed,while, at this stage, a filamentous staining reaction couldbe seen with the desmin antibodies. During furtherdevelopment, cross-striation was detected within myo-cardial cells using the polyclonal titin antibody from 9.0d.p.c. onwards, i.e. before such striation could bedetected with the desmin antibodies.

From these data, we conclude that titin synthesis mayanticipate desmin expression in the developing mousemyocard, although the level of expression of the formerprotein remains low until 9.0 d.p.c.

Key words: desmin, titin, mouse cardiogenesis, heartmuscle, myotome, keratin, vimentin, fibronectin.

Introduction

The protein constituents of intermediate-sized fila-ments of the cytoskeleton in cells of adult animals havebeen described to be expressed in a more or less tissue-specific fashion (Quinlan et al. 1985). Keratin normallyoccurs only in epithelial tissues, while neurofilamentsand glial fibrillary acidic protein (GFAP) are mainlyrestricted to neural cell types (Debus et al. 1983a; Shawet al. 1986; for review see Holtzer et al. 1982). Desminhas so far been found to be virtually muscle specific(Debus et al. 1983b; for exceptions see Molengraft et al.1986 and Franke and Moll, 1987), while vimentin occursin cells of mesenchymal origin, but is also occasionallycoexpressed with the other intermediate filament pro-teins (IFP) (Franke et al. 1982). The expression of IFPin developing embryos has been studied by severalinvestigators (reviewed by Traub, 1985). From theseresults, it has become obvious that, apart from thelamins, cytokeratins are the first cytoplasmic IFP to be

expressed in early embryogenesis (Jackson et al. 1981).Embryonal cells containing this type of IFP are multi-potential in nature and switch on the expression ofvimentin when differentiating into mesenchymal cells(Franke et al. 1982; Traub, 1985). Subsequently, differ-entiation into mature cells results in the expression ofthe more specific IFP, such as desmin in muscle cellsand GFAP in glial cells. However, coexpression ofvimentin with these IFP is seen during embryogenesisand even after birth (Osborn et al. 1981; Lazarides et al.1982; Schnitzer et al. 1981). During myogenesis vimen-tin and desmin are expressed in sequence in differen-tiating myocytes (Fischman, 1986). In replicating myo-blasts, vimentin is found in early stages of developmentat which desmin is undetectable. When the myogeniccells withdraw from the cell cycle at an early stage ofmyofibrillogenesis, desmin expression is initiated(Fischman, 1986). As muscle cell differentiation pro-ceeds, desmin expression becomes more prominent,while its localization becomes more and more restricted

586 G. Schaart and others

to the outer circumference of the Z-band, both inskeletal and cardiac striated muscle cells (Fischman,1986; Hill etal. 1986; Danto and Fischman, 1984). Mostof the work on IFP expression in muscle morphogenesishas concentrated on avian tissues (Tapscott et al. 1981;Bennett et al. 1981; Tokuyasu et al. 1984), whilemammalian myofibrillogenesis has hardly been studiedat this level. Since fundamental differences have, how-ever, been noted during myogenesis in avians andmammals (Kaufman and Navaratnam, 1981; Bignami etal. 1984), we have studied desmin and vimentin ex-pression in mouse embryos at 8.0 to 9.5 days postcoitum (d.p.c). It was postulated that during avian invivo and in vitro myofibrillogenesis desmin is one of theearliest known markers for cells in the myogenic lineageand that proteins such as titin and myosin heavy chainoccur later. We also studied the development of titin,which has been identified as a specific component incardiac and skeletal muscle (Maruyama et al. 1976;Wang etal. 1979; Maruyama, 1986).

Materials and methods

EmbryosThe embryos used for this study were from spontaneousmatings of mice of the C75bl/CBA (BCBA) strain. Thepresence of a vaginal plug indicated a successful mating. Theconceptuses were thus presumed to be 0.5 days old at noon(12:00) on the day the vaginal plug was found. At 8.0, 8.25,8.5, 9.0 and 9.5 days post coitum (d.p.c.) embryos weredissected from the uterus and washed in cold phosphate-buffered saline (PBS; 4°C). Embryos were quickly frozen inliquid N2 after removal from the uterus and immersed inTissue Tek. The developmental stages are designated accord-ing to Theiler (1972).

Indirect immunofluorescence microscopy5 j«n thick cryostat sections were air dried, fixed with meth-anol at -20cC for 5min, followed by acetone fixation at—20°C for lmin. After air drying for 15min at roomtemperature, the sections were incubated with the primaryantibody for 30min at room temperature, and washed threetimes (5 min each) in PBS. Then they were incubated with thesecondary antibody for 30 min at room temperature and againwashed three times (5min each) in PBS, for 5min in distilledwater and finally for 5 min in methanol. Sections weremounted in Mowiol (Hoechst, Frankfurt, FRG) containing2.5% (w/v) NaN3 to retard fading (Johnson and Davidson,1982). Slides were examined using a Zeiss Universal micro-scope equipped with epi-illumination optics.

Gel electrophoresis and immunoblottingCytoskeleton preparations from 9.5 d.p.c. mouse embryoswere made as follows. After dissection of the embryos fromthe uterus, a high-salt buffer (1.5M-KC1, 0.5% Triton X-100,5mM-EDTA, 0.4mM-phenylmethylsulphonylfluoride and10mM-Tris-HG, pH7.2) extraction was performed for 5 minat 4°C by homogenization of the embryo in a Dounce potter.The pellet obtained by centrifugation for 5 min at 12 000 g waswashed with cold (4°C) low-salt buffer (5 mM-EDTA, 0.4 ITIM-phenylmethylsulphonylfluoride and 10mM-Tris-HCl,pH 7.2), essentially as described by Moll et al. (1982). After asecond washing step in PBS, the cytoskeleton preparation was

dissolved by boiling for 5 min in SDS-sample buffer(Laemmli, 1970).

One-dimensional gel electrophoresis was performed in10% polyacrylamide slab gels containing 0.1% SDS(Laemmli, 1970). To compare the amounts of protein loadedon each lane, gels were stained with Coomassie Brilliant BlueR250 (Gurr, Hopkin and Williams, Chadwell Heath, Essex,UK) as described before (Broers et al. 1986). Two-dimen-sional gel electrophoresis was performed essentially as de-scribed by O'Farrell (1975). In the first dimension, isoelectricfocusing was performed in 4 % polyacrylamide (Biorad Lab-oratories, California, USA) rod gels containing 1% Biolyte,pH3.5-10 (Biorad Laboratories, California, USA). For thesecond dimension, the rod gels were applied directly onto thestacking gel of SDS-polyacrylamide gels.

For immunoblotting experiments, the electrophoreticallyseparated polypeptides were transferred to a nitrocellulosesheet (Schleicher and Schiill Membrane Filters BA 85, Das-sel, FRG) by blotting for one hour at 100 V in a cold (4°C)buffer containing 25mM-Tris-HCl, 192mM-glycine, 0.02%SDS and 20% methanol (pH8.3) (Towbin etal. 1979). Theblots were incubated for 90 min with PBS containing 0.05%Tween 20 (Sigma). All reagents were diluted in this solution,which was also used for the washing steps. After incubationovernight with undiluted culture supernatants of the primaryantibodies RD301, RV202, CK18-2 or BV1118, the blotswere washed three times for 10 min and incubated for 1 h withperoxidase conjugated rabbit anti-mouse IgG (DAKOpatts,Glostrup, Denmark) diluted 1:400. The blots were washedagain three times for 10 min in 0.05% Tween 20/PBS andonce in PBS alone for 10 min and stained with4-chloro-l-naphtol (Merck, Darmstadt, FRG) and 0.12%hydrogen peroxide (Merck, Darmstadt, FRG). After stainingthe blots were rinsed for 5 min with water.

AntibodiesThe following antibody preparations were used in this study:

1. A polyclonal antiserum to chicken gizzard desmin(pDes). Preparation and characterization of this rabbit anti-serum have been described elsewhere (Ramaekers et al.1985). For indirect immunofluorescence microscopy, thisserum was diluted 1:50 in PBS.

2. Two mouse monoclonal antibodies to desmin (DEB5and DER11; DAKOpatts, Glostrup, Denmark; Debus et al.19836). These antibodies were used as undiluted culturesupernatants in the immunohistochemical assays.

3. A mouse monoclonal antiserum to desmin (RD301),giving a weak, although specific, reaction on muscle cells inadult and embryonic mouse when used in the indirect immu-nofluorescence assay, was used for the immunoblotting as-says. This antibody has been described before (Pieper et al.1989).

4. An affinity-purified polyclonal antiserum to bovine lensvimentin (pVim). Purification and characterization of thispolyclonal rabbit antiserum have been described in detailelsewhere (Ramaekers et al. 1983).

5. A mouse monoclonal antibody (BV1118) of the IgMsubclass, reactive with vimentin of human, bovine, rabbit,hamster, mouse and chicken origin (C. Viebahn, unpublisheddata). This antibody, when tested in the indirect immunoflu-orescence assay, stains tissues of mesenchymal origin such asfibroblasts, endothelial cells, some smooth muscle cells,glomeruli in the kidney, but does not stain most adultepithelial cells. In cultured cells (BHK, HeLa), a filamentousstaining pattern is obtained with antibody BV1118. Nosignificant reaction is found in cultured cells such as T24, RT4

Desmin and titin in mouse embryos 587

1AB CD EF

Fig. 1. Characterization of monoclonal antiserum BV1118 by one- (A, C, E) and two-dimensional (B, D, F) immunoblottingon cytoskeletal preparations of bovine lens (A, B) and BHK-cells (C-F). Note that antiserum BV1118 reacts exclusivelywith vimentin (v) and its breakdown products (A-D), both from calf and hamster. Reincubation of the immunoblots withthe monoclonal desmin (d) antiserum (RD301) (E, F) confirms the vimentin-specific reaction of antiserum BV1118.

and MCF-7, known to be vimentin negative (Heuijerjans etal.1989). In one- and two-dimensional immunoblotting assays ofcytoskeletal extracts from bovine lens, BHK (Fig. 1), andHela cells, this monoclonal antibody shows a positive reactionwith a protein band migrating at the molecular weight leveland isoelectric pH of vimentin. This antibody was used asundiluted culture supernatant in the immunohistochemicalassays.

6. A mouse monoclonal antiserum to vimentin (RV202),giving a weak, although specific, reaction on stromal cells andother mesenchymal tissues in adult and embryonic mouse inthe indirect immunofluorescence assay, was used for immuno-blotting experiments. This antibody has been described indetail elsewhere (Ramaekers et al. 1987; Viebahn et al. 1988;Pieper etal. 1989).

7. An affinity-purified polyclonal antiserum to human skinkeratins (pKer). This rabbit antiserum is described elsewhere(Ramaekers et al. 1983). For indirect immunofluorescencemicroscopy, this antiserum was diluted 1:10 in PBS.

8. A mouse monoclonal antibody (CK18-2) specificallyrecognizing cytokeratin 18 was raised against human meso-thelial cells. This antibody was used as undiluted culturesupernatant (Broers et al. 1986).

9. An affinity-purified polyclonal rabbit antiserum to titin,isolated from Physarum polycephalum (Kind gift from Dr D.Gassner, Bonn, FRG), was used in a 1:25 dilution. Prep-aration and specificity of this antiserum have been describedby Gassner (1986). When tested on adult mouse tissues usingthe indirect immunofluorescence assay, this antiserum reactedspecifically with striated (skeletal- and cardiac-) muscle cells.No reaction was found in smooth muscle cells or in nonmusclecells.

10. A polyclonal antiserum against human fibronectin waspurchased from DAKOpatts (Glostrup, Denmark) and usedat a dilution of 1:120.

As secondary antibodies fluoresceine (FITC)-conjugatedgoat anti-mouse IgG, goat anti-mouse IgM and goat anti-rabbit IgG were used in a dilution of 1:60. All FITC-conjugated antisera were obtained from Nordic Immuno-chemicals (Tilburg, The Netherlands).

In order to prove that the correct protein constituents arerecognized in mouse embryos, studies were performed with9.5 d.p.c. embryos. Fig. 2 shows that with the monoclonalantisera to vimentin, desmin and keratin 18, these individualcomponents can be detected in cytoskeletal preparations from9.5 d.p.c. embryos.

- d d -

—k

2A B D

Fig. 2. Immunoblotting study on cytoskeletal preparationsfrom 9.5 d.p.c. mouse embryos (lanes A-D). Immunoblotswere incubated as follows: lane A with the monoclonaldesmin antiserum (RD301); lane B with the monoclonalvimentin antiserum (RV202); lane C with RD301 andsubsequently with RV202; lane D as lane C, but thereafterincubated with the monoclonal keratin 18 antiserum(CK18-2). For comparison, lane E depicts an immunoblotof a chicken gizzard desmin preparation incubated withRD301 and lane F a cytoskeletal preparation of cell lineRT4 incubated with CK18-2, to show the position ofdesmin (d) and keratin 18 (k), respectively, v, vimentin.

Results

The results obtained in the immunohistochemicalstudies, performed on mouse embryos of 8.0, 8.25, 8.5,9.0, and 9.5 d.p.c, are summarized in Table 1 anddepicted in Figs 3-9.

Presomite embryo (Theiler stage 11)At 8.0 d.p.c, the polyclonal antiserum pKer shows astrongly positive reaction in the visceral endoderm. Anextremely intense reaction with this antiserum was alsoobserved in the parietal endoderm, while the mesodermand ectoderm were virtually negative. AntibodyCK18-2 reacted in a similar fashion but weaker. Thevimentin antiserum (pVim) showed a pronounced reac-tion in the mesoderm, which was particularly obvious in


the tangentially sectioned areas of the embryos. Thedesmin polyclonal antiserum was negative in all embry-onic tissues, but did stain the extraembryonic smoothmuscle tissue of the uterus. Fibronectin was detectedextracellularly in the embryonic mesoderm and innonembryonic tissues, but not in the visceral endodermand ectoderm. The polyclonal titin antiserum (pTitin)was also tested at 8.0 d.p.c. and found to be negative.

Heart rudiment (Theiler stage 12)In the mouse, heart development starts in the midline asan unpaired anlage (Kaufman and Navaratnam, 1981)rostral from the neural plate and caudal from theembryonic-extraembryonic junction (Theiler, 1972).This early stage of heart formation was studied in a 8.25d.p.c. embryo (Fig. 3) in which the heart-formingregion can be seen to have bent ventrally in order toform the foregut pocket. In the sagittal sections throughthis region, we have observed no reaction, or at best anextremely weak staining reaction, with the desminpolyclonal antiserum (pDes) (see Fig. 3A) However,the basal plate of the neuroectoderm overlying theforegut pocket dorsally showed a positive stainingreaction with the desmin antiserum while the mesodermand endoderm were negative. Using a titin polyclonalantiserum (pTitin), single positive dots were observedin the heart rudiment (Fig. 3B). The mesoderm, theectoderm, the endoderm and the foregut pocket werenegative. The vimentin monoclonal antiserum(BV1118) (Fig. 3C) and the polyclonal antiserumagainst vimentin were positive in the mesoderm and theneuroectoderm as well as in the heart anlage. Inparticular with the monoclonal vimentin antiserum(Fig. 3C), a strong filamentous staining was found inthe heart anlage. Note that there is also a strongpositive staining reaction in the neuroectoderm over-lying the foregut pocket, indicating coexpression withdesmin in these cells. An intense reaction with themonoclonal antibody against cytokeratin 18 (CK18-2)(Fig. 3D) was observed in the visceral endoderm, theforegut pocket and the visceral layer of the yolk sac atthis stage. Except for the amnion epithelium, bothectoderm and neuroectoderm are still negative for themonoclonal and polyclonal keratin antibodies. How-ever, mesodermal cells anterior to, and in, the heartanlage show a distinct positive reaction for keratin(Fig. 3D). Fibronectin showed the outlines of the base-ment membrane of the endoderm and the amnionepithelium, and was detected in the mesenchyme

(Fig. 3E), as well as in the heart anlage. The endodermand ectoderm are negative with this antiserum.

A slightly more advanced stage of heart developmentis shown in Fig. 4 in horizontal sections of a 8.5 d.p.c.mouse embryo. Cells of the presumptive endocard cannow be seen between the endoderm and the developingmyocard. Desmin is now exclusively observed in thedeveloping heart (Fig. 4A), and could not be detectedin the neuroectoderm or any other region of theembryo. Titin could be detected in the developing heartregion, again in a punctate distribution (Fig. 4B).Vimentin was coexpressed with desmin in this region,and showed a strong filamentous immunofluorescencestaining reaction in the myocardial cells (Fig. 4C), butvirtually no reaction in the endoderm. Underlying theendoderm, a few vimentin positive, but desmin nega-tive, cells can be identified as the first endocardial cells.The neuroectoderm was still positive for vimentin(results not shown). Both the polyclonal and mono-clonal keratin antisera stained cells of the heart anlage(Fig. 4D) as well as the overlying endoderm, while theneuroectoderm was negative.

Tubular heart (Theiler stage 13)The next stage during heart development is the forma-tion of a tubular heart in which the endocard is almostcompletely surrounded by myocard. Fig. 5 showsoblique sections through such a 8.5 d.p.c. embryonicheart. The formerly punctate staining pattern of titinantibodies is now replaced by a more filamentousstaining pattern (Fig. 5B). The desmin-staining reac-tion remains filamentous, comparable to the foregoingdevelopmental stage (Fig. 5A). With the monoclonaland polyclonal vimentin antisera (Fig. 5C), a strongpositive reaction was observed in the myocard and theendocard. Also with the keratin antisera we have founda positive reaction in the myocard of the tubular heart(result not shown).

Segmented heart (Theiler stage 14)At this stage, the anlagen of the different parts of theheart can be distinguished, i.e. the anlagen of theventricular and the atrial compartments (Fig. 6). As in8.5-day-old embryos, in 9.0 d.p.c. mouse embryosdesmin expression seems to be restricted to the hearttissue, except for the myotomes (see below). The cellsforming the myocardial wall are strongly positive withthe polyclonal desmin antiserum (Fig. 6A), while theendocardial tissues are distinctively negative (asterisksin Fig. 6A). The monoclonal desmin antibody DEB5

Table 1. Early expression of desmin and titin in the developing heart of mouse embryosStages of cardiogenesis(Theiler stage)

Heart rudiment8.25 d.p.c. (12)

Heart rudiment8.5 d.p.c. (12)

Tubular heart8.5 d.p.c. (13)

Segmented heart9.0d.p.c. (14)

Segmented heart9.5 d.p.c. (14)

desmin expression• pattern

titin expressionpattern dots

filaments

dots

filaments

filaments

filaments

filaments andstriation




Fig. 3. Immunofluorescence micrographs of sagittal frozen sections from 8.25 d.p.c. embryos of the mouse (Theiler stage 12)showing the heart rudiment (HR), the foregut pocket (FP) and the neuroectoderm (NE). The sections were incubated with:(A) the polyclonal antiserum against desmin (pDes); (B) the polyclonal antiserum against titin (pTitin); (C) the monoclonalantiserum BV1118 against vimentin; (D) the monoclonal antiserum CK18-2 against cytokeratin 18 and (E) the polyclonalantiserum against fibronectin (pFN). Bars indicate 25 fim.

590 G. Schaaft and others

showed only a partial reaction in the myocardial wall(results not shown). An extensive staining reaction wasfound in all cells of the myocardial wall with the titinantiserum (Fig. 6B), which was negative in the endocar-dial cells. In some of the myocardial cells, a transverse

Fig. 4. Immunofluorescence micrographs of horizontalsections from 8.5 d.p.c. mouse embryos (Theiler stage 12),showing the heart rudiment (HR) and the endocard (Ec).Sections were incubated with (A) the polyclonal desminantiserum (pDes); (B) the polyclonal titin antiserum(pTitin); (C) the polyclonal vimentin antiserum (pVim), and(D) the polyclonal keratin antiserum (pKer). Bar indicates25 /nn.

cross-striation was found (Fig. 7B) with this polyclonaltitin antiserum, while there was no cross-striation foundwith the desmin (Fig. 7A) and the vimentin antibodiesat this stage. The endocardial cells as well as themyocardial cells are specifically stained with the vimen-tin antiserum (Fig. 6C). Keratin expression seems to bedrastically reduced in the heart anlage at this stage,although part of the cells still showed a weak punctateto fibrillar staining reaction with the monoclonal keratinantiserum CK18-2 (Fig. 6D). Keratin-positive reac-tions of differentiating pericardial cells are also ob-served in the primitive oral epithelium, the thyroidanlage, the lateral plate of the mesoderm and theepidermal ectoderm with the monoclonal keratin anti-body (CK18-2). During further differentiation of myo-cardial cells (e.g. in 9.5 d.p.c. embryos), coexpressionof desmin and vimentin is evident. With both antibodiescross-striation can now be observed in individual cells atthis stage (Fig. 8A, B) in addition to the cross-striationsseen with the titin antibody. Keratin is still weaklyexpressed in some myocardial cells of 9.5 d.p.c. mouse

n embryos (results not shown).

MyotomeIn the mouse, somites begin to form at Theiler's stage12 (Theiler, 1972). The somites at this stage are dis-tinctly negative for desmin, titin and keratin, butpositive for vimentin. Differentiation of the somite intodermatome, myotome and sclerotome starts at day 9.0d.p.c. (Ede and El-Gadi, 1986; Theiler stage 14) andthis is accompanied by a positive staining reaction withthe desmin and titin antisera in the most cranialmyotomes of the embryo (Fig. 9). Desmin stainingshows a filamentous staining pattern (Fig. 9A), whiletitin antibodies give rise to both a punctate and afilamentous staining pattern (Fig. 9B). The vimentinantibodies stain all three somite-derived tissues (derma-tome, myotome and sclerotome) (Fig. 9C), whereasthese tissues are distinctly negative with all the keratinantibodies tested in this study (Fig. 9D).

Discussion

Desmin has been described to be a muscle-specificintermediate filament (IF) component in adult ver-tebrates (Hill etal. 1986), while titin is a marker in adultstriated muscle (Wang et al. 1979). Both componentsmay also serve as early markers of the anlagen of thesetissues during embryogenesis (Tokuyasu et al. 1984;Tokuyasu and Maher, 1987). In early stages of mam-malian myofibrillogenesis, however, the intermediate


Fig. 5. Immunofluorescence micrographs of frozen sections from 8.5 d.p.c mouse embryos (TheOer stage 13), showing thetubular heart. Sections were incubated with (A) the monoclonal desmin antiserum (DEB5); (B) the polyclonal titinantiserum (pTitin) and (C) the monoclonal vimentin antiserum (BV1118). Bar indicates 50/im.

filament protein (IFP) vimentin is expressed in thedeveloping muscle cells, either without or in combi-nation with desmin. Van Muijen et al. (1987) and Kurucand Franke (1988) have recently shown that humanmyocardial cells may even coexpress three differenttypes of IF-proteins, i.e. keratins, vimentin and desmin(see also Huitfeldt and Brandtzaeg, 1985).

The main studies, so far, on the expression of muscle-specific components during embryogenesis have con-centrated on the chicken system (for a review seeFischman, 1986). Recently, Tokuyasu and Maher(1987) have described the distribution of titin in chickencardiac premyofibril stages. Hill et al. (1986) studied theinteraction between titin and desmin in postmitoticmononucleated myoblast and concluded that the spatialorganization of both components was not coupled. To amuch lesser extent, such experiments have been per-formed in the mouse system (for an example, seeSassoon et al. 1988). Studies on the formation of IFcomponents during early mouse embryogenesis byJackson era/. (1980,1981) and Franke etal. (1982) haveshown that no desmin expression can be detectedbefore day 8.0 d.p.c.

In the present study, a punctate staining pattern oftitin antibodies was found as the earliest sign of myo-genic differentiation in the mouse heart at Theiler'sstage 12 (8.25 d.p.c). Still at the same stage, only a fewhours later, desmin expression was also initiated in theheart rudiment, showing a filamentous staining pattern,while titin still showed a punctate staining pattern.Again a few hours later when the heart changed to atubular organization at Theiler's stage 13, the titinpattern changed to a fibrillar intracellular distribution

with the desmin-staining pattern remaining the same asin the foregoing stage. Striation of titin became appar-ent in the segmented heart at 9.0 d.p.c. (Theiler stage14). A few hours later at 9.5 d.p.c, but still the samestage according to Theiler, cross-striation of desmin andvimentin was observed. Myogenesis in somites (i.e. inthe myotome) was similarly accompanied by a punctatetitin staining pattern (Theiler stage 14) which wasfollowed by a change to a fibrillar-staining pattern. Buthere a delay between the appearance of titin anddesmin could not be detected, possibly due to the speedof differentiation in myotome cells.

To our knowledge, this is the first description of theinitiation of desmin expression in the vertebrate heart.Later stages of heart development have been describedby Kuruc et al. (1988) in several species. Titin ex-pression, however, has been described in the earlystages of chick heart development by Tokuyasu et al.(1988), who also found a punctate staining pattern inthe first stages. During further differentiation, thispattern changes into a fibrillar staining reaction.

In the underlying study, it became also evident thatthe first expression of mouse desmin could be noted inthe neuroectoderm of 8.25 d.p.c. mouse embryos. At8.5 d.p.c. and later stages, the neuroectodermal layerwas negative for desmin.

Coexpression of different types of IFP is a commonfeature in developing embryonic tissues, and to someextent also in certain adult organs (reviewed by Traub,1985; Viebahn etal. 1987, 1988). Recently, Van Muijenet al. (1987) and Gown et al. (1988) demonstrated atriple expression of keratins, vimentin and desminin human fetal heart muscle cells. In mouse


Fig. 6. Immunofluorescence micrographs of frozen sections from 9.0 d.p.c. mouse embryos (Theiler stage 14), showing theneural tube (NT), thyroid anlage (TA), ventricular compartments (V), atrial compartments (A), and endocard (asterisks),incubated with (A) the polyclonal desmin antiserum (pDes); (B) the polyclonal titin antiserum (pTitin); (C) the monoclonalvimentin antiserum (BV1118); (D) the monoclonal keratin 18 antiserum (CK18-2). Bar indicates 100/im.


Fig. 7. Immunofluorescence micrographs of a doublestaining of embryonic mouse myocardium (Theiler stage 14,9.0 d.p.c), using (A) a monoclonal desmin antibody(DER11) and (B) the polyclonal titin antibody (pTitin).Note the cross-striation in the titin-staining reaction (arrowin B) and lack of obvious cross-striation at correspondingsites in the desmin staining (A). Bar indicates 6 nm.

embryos, vimentin and keratin 18 were coexpressed inthe differentiating myocard together with titin at first,and later also with titin and desmin. The endocardialcells were always stained by the vimentin antibodiesonly. Desmin and keratin coexpression was also foundto be a transient feature in heart development by Kurucet al. (1988). At 9.0 d.p.c, keratin reactivity in themyocard was drastically decreased, resulting in a nega-tive reaction in 9.5 d.p.c. mouse embryos. However, atthis stage, vimentin and desmin are still coexpressedand even colocalized as concluded from their cross-striated immunofluorescence pattern.

Although in vitro studies of myogenesis show thatdesmin is a candidate for the first sign of myogenicdifferentiation, our studies clearly show that, in vivo,titin, and not desmin, is the first muscle-specific proteinto be expressed in presumptive myogenic cells (Hill etal. 1986; Sassoon et al. 1988). The titin antiserum usedin this study showed a specific and exclusive immuno-reactivity in the heart anlage of the mouse embryobefore desmin expression could be detected in thisregion, at 8.25 d.p.c. (Theiler stage 12). The typicalpunctate feature of this labeling pattern, which iscomparable to that described by Tokuyasu and Maher(1987) for early stages of cardiac myofibrillogenesis inchick embryos, supports the assumption that, at thisstage, the antiserum does indeed react with titin.

Unfortunately, no immunochemical proof can be

Fig. 8. Immunofluorescencemicrographs of frozen sections throughthe heart of a 9.5 d.p.c. mouse embryo(Theiler stage 14). Sections wereincubated with (A) the polyclonaldesmin antiserum (pDes) and (B) thepolyclonal vimentin antiserum (pVim).Arrows show cross-striations inmyocardial cells. Bar indicates 9/OTI.


Fig. 9. lmmunofluorescence micrographs offrozen sections of the myotome of a 9.5 d.p.c.mouse embryo (Theiler stage 14). Sections wereincubated with (A) a polyclonal desminantiserum (pDes), (B) a polyclonal titinantiserum (pTitin), (C) a monoclonal vimentinantiserum (BV1118), and (D) a polyclonalkeratin antiserum (pKer). (ep, epidermal anlage;d, dermatome; m, myotome;s, sclerotome). Barindicates 20/an.

obtained for this observation, since the concentration ofthe antigen in 8.25 d.p.c. embryos is far too low to allowits detection in immunoblotting studies. At stage 8.5d.p.c, titin is still present as spots in the developingmyocard, while desmin can now clearly be detected andoccurs in a filamentous fashion inside these premyocar-dial cells. Desmin and titin were coexpressed in 9.0d.p.c. embryonic heart and myotome and showedsimilarly strong intensities in the immunofluorescenceassays on frozen sections of this tissue. At this stage,however, a number of cells showed a striated titin

staining pattern, while we could not observe such anorganization for desmin. At 9.5 d.p.c, desmin, titin andvimentin were found to be colocalized in these cross-striations. Although it should be kept in mind that thestudies so far have been performed in different systems,our observations that titin expression anticipates des-min synthesis seem in contrast with the in vitro findingsof Hill et al. (1986). These authors showed desminexpression in presumptive replicating myoblasts pres-ent in embryonic chick skeletal muscle cultures. Theycould not detect titin until the postmitotic mononu-


cleated myoblast stage and therefore suggested that thisconstituent is expressed later than desmin in the courseof skeletal muscle myofibrillogenesis. Apparently, des-min and titin expression are differently coordinated invivo and in vitro.

As far as the myotome is concerned, the presentstudy shows almost simultaneous appearance of titinand desmin in nascent myotome cells. This establishestitin as an early myogenic marker in the myotome inaddition to desmin which was hitherto thought to be theearliest marker of myotomal differentiation (Solurshand Meier, 1986; Kaehn et al. 1988; Viebahn, 1989).Furthermore, the characteristic developmental changeof the intracellular distribution of titin from punctate tofibrillar is seen in the myotome as well as in the heart,suggesting similar sequences of early myogenic differ-entiation operating both organs.

We are grateful to Annemiete van de Kemp for technicalassistance.

References

BENNETT, G. S., TAPSCOTT, S. J., KLEINBART, F. A., ANTIN, P. B.

AND HOLTZER, H. (1981). Different proteins associated with 10-nanometer filaments in cultured chick neurons and nonneuralcells. Science 212, 567-569.

BIGNAMI, A. AND DAHL, D. (1984). Early appearance of desmin,the muscle-type intermediate filament protein, in the rat embryo./. Histochem. Cytochem. 32, 473-476.

BROERS, J. L. V., CARNEY, D. N., KLEIN ROT, M., SCHAART, G.,

LANE, E. B., VOOUS, G. P. AND RAMAEKERS, F. C. S. (1986).

Intermediate filament proteins in classic and variant types ofsmall cell lung carcinoma cell lines: a biochemical andimmunochemical analysis using a panel of monoclonal andpolyclonal antibodies. / . Cell Sci. 83, 37-60.

DANTO, B. I. AND FISCHMAN, D. A. (1984). Lmmunocytochemicalanalysis of intermediate filaments in embryonic heart cells withmonoclonal antibodies to desmin. / . Cell Biol. 98, 2179-2191.

DEBUS, E., WEBER, K. AND OSBORN, M. (1983a). Monoclonal

antibodies specific for glial fibrillary acidic (GFA) protein and foreach of the neurofilament triplet polypeptides. Differentiation 25,193-203.

DEBUS, E., WEBER, K. AND OSBORN, M. (1983fc). Monoclonal

antibodies to desmin, the muscle specific intermediate filament.EMBO J. 2, 2305-2312.

EDE, D. A. AND EL-GADI, A. D. A. (1986). Genetic modificationsof developmental acts in chick and mouse somite development.In Somites in Developing Embryos (ed. R. Bellairs, D. A. Edeand J. W. Lash) Plenum Press, New York, pp.209-224.

FISCHMAN, D. A. (1986). Myofibrillogenesis and the morphogenesisof skeletal muscle. In Myology: Basic and Clinical, (ed.A. G. Engel and B. Q. Baker) McGraw-Hill, New York,pp.5-30.

FRANKE, W. W., GRUND, C , KUHN, C , JACKSON, B. W. AND

ILLMENSEE, K. (1982). Formation of cytoskeletal elements duringmouse embryogenesis. III. Primary mesenchymal cells and thefirst appearance of vimentin filaments. Differentiation 23, 43-59.

FRANKE, W. W. AND MOLL, R. (1987). Cytoskeletal components oflymphoid organs. I. Synthesis of cytokeratins 8 and 18 anddesmin in subpopulations of extrafollicular reticulum cells ofhuman lymph nodes, tonsils and spleen. Differentiation 36,145-163.

GASSNER, D. (1986). Myofibrillar interaction of blotimmunoaffinity-purined antibodies against native titin as studiedby indirect immunofluorescence and immunogold staining. Eur.J. Cell Biol. 40, 176-184.

GOWN, A. M., BOYD, H. C , CHANG, Y., FERGUSON, M., REICHLER,

B. AND TIPPENS, D. (1988). Smooth muscle cells can express

cytokeratins of "simple" epithelium. lmmunocytochemical andbiochemical studies in vitro and in vivo. Am. J. Path. 132,223-232.

HEUUERJANS, J., PIEPER, F. R., RAMAEKERS, F. C. S., TIMMERMANS,

L. J. M., KUUPERS, H. J. H., BLOEMENDAL, H. AND VENROOU

VAN, W. J. (1989). Association of mRNA and eIF-2a'with thecytoskeleton in cells lacking vimentin. Expl Cell Res. 181,317-330.

HILL, C. S., DURAN, S., ZHONGXIANG, L., WEBER, K. AND

HOLTZER, H. (1986). Titin and myosin, but not desmin, arelinked during myofibrillogenesis in postmitotic mononucleatedmyoblasts. / . Cell Biol. 103, 2185-2196.

HOLTZER, H., BENNETT, G. S., TAPSCOTT, S. J., CROOP, J. M. AND

TOYAMA, Y. (1982). Intermediate-sized filaments: changes insynthesis and distributions in cells of the myogenic andneurogenic lineages. Cold Spring Harbor Symp. quant. Biol. 46,317-326.

HUTTFELDT, H. S. AND BRANDTZAEG, P. (1985). Various keratinantibodies produce immunohistochemical staining of humanmyocardium and myometrium. Histochemistry 83, 381-389.

JACKSON, B. W., GRUND, C , SCHMID, E., B U R H , K., FRANKE, W.

W. AND ILLMENSEE, K. (1980). Formation of cytoskeletalelements during mouse embryogenesis. I. Intermediate filamentsof the cytokeratin type and desmosomes in preimplantationembryos. Differentiation 17, 161-179.

JACKSON, B. W., GRUND, C , WINTER, S., FRANKE, W. W. AND

ILLMENSEE, K. (1981). Formation of cytoskeletal elements duringmouse embryogenesis. II. Epithelial differentiation andintermediate-sized filaments in early postimplantation embryos.Differentiation 20, 203-216.

JOHNSON, G. D. AND DAVIDSON, R. S. (1982). Fading ofimmunofluorescence during microscopy. A study of thephenomenon and its remedy. J. Immun. Methods 55, 231-242.

KAEHN, K., JACOB, H. J., CHRIST, B., HINRICHSEN, K. AND

POELMANN, R. E. (1988). The onset of myotome formation in thechick. Anat. Embryol. 177, 191-201.

KAUFMAN, M. H. AND NAVARATNAM, V. (1981). Earlydifferentiation of the heart in mouse embryos. / . Anat. 133,235-246.

KURUC, K. AND FRANKE, W. W. (1988). Transient coexpression ofdesmin and cytokeratins 8 and 18 in developing myocardial cellsof some vertebrate species. Differentiation 38, 177-193.

LAEMMLI, U. K. (1970). Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature, Lond. 2X1,680-685.

LAZAIUDES, E., GRANGER, B. L., O'CONNOR, C. M., BRECKLER, J.,

PRICE, M. AND DANTO, S. I. (1982). Desmin- and vimentin-containing filaments and their role in the assembly of the Z-diskin muscle cells. Cold Spring Harbor Symp. quant. Biol. 46,351-378.

MARUYAMA, K. (1986). Connectin, an elastic filamentous protein ofstriated muscle. Int. Rev. Cytol. 104, 81-114.

MARUYAMA, K., NATORI, R. AND NONOMURA, Y. (1976). New elastic

protein from muscle. Nature, Lond. 262, 58-60.MOLENGRAFT VAN DE, F . , RAMAEKERS, F . , JAP, P . , VOOUS, P . AND

MUNGYER, G. (1986). Changing intermediate-sized filamentpatterns in metastatic hepatocellular carcinoma cells of theguinea pig. Virchows Arch. B 51, 285-301.

MOLL, R., FRANKE, W. W., SCHILLER, D. L., GEIGER, B. AND

KREPLER, R. (1982). The catalog of human cytokeratins: patternsof expression in normal epithelia, tumors and cultured cells. Cell31, 11-24.

MUTJENVAN, G. N. P., RUTTER, D. J. AND WARNAAR, S. O. (1987).

Coexpression of intermediate filament polypeptides in humanfetal and adult tissues. Lab. Invest. 57, 359-369.

O'FARRELL, P. H. (1975). High resolution two-dimensionalelectrophoresis of proteins. / . biol. Chem. 230, 4007-4021.

OSBORN, M., LUDWIG-FESTL, M., WEBER, K., BIGNAMI, D., DAHL,

D. AND BAYREUTHER, K. (1981). Expression of glial and vimentintype intermediate filaments in cultures derived from human glialmaterial. Differentiation 19, 161-167.

PIEPER, F. P., SCHAART, G., KRIMPENFORT, P. J., HENDERIK, J. B.,

MOSHAGE, H. J., RAMAEKERS, F. C. S., BERNS, A. AND

BLOEMENDAL, H. (1989). Transgenic expression of the muscle-


specific intermediate filament protein desmin in non-muscle cells.J. Cell Biol. 108, 1009-1024.

QUINLAN, R. A., SCHILLER, D. L., HATZFELD, M., ACHTSTATTER,T , MOLL, R., JORCANO, J. L., MAGIN, T. M. AND FRANKE, W.W. (1985). Patterns of expression and organization of cytokeratinintermediate filaments. Ann. N.Y. Acad. Sci. 455, 282-306.

RAMAEKERS, F. C. S., HUYSMANS, A., SCHAART, G., MOESKER, O.AND Voous, G. P. (1987). Tissue distribution of keratin 7 asmonitored by a monoclonal antibody. Expl Cell Res. 170,235-249.

RAMAEKERS, F. C. S., MOESKER, O., HUYSMANS, A., SCHAART, G.,WESTERHOF, G., WAGENAAR, SJ. SC., HERMAN, C. J. AND VOOUS,G. P. (1985). Intermediate filament proteins in the study ofrumor heterogeneity: an in-depth study on rumors of the unnaryand respiratory tracts. Ann. N.Y. Acad. Sci. 455, 614-634.

RAMAEKERS, F. C. S., PUTS, J. J. G., MOESKER, O., KANT, A.,HUYSMANS, A., HAAG, D., JAP, P. H. K., HERMAN, C. J. ANDVOOLTS, G. P. (1983). Antibodies to intermediate filamentproteins in the immunohistochemical identification of humantumours: an overview. H'tstochemical J. 15, 691-713.

SASSOON, D. A., GARNER, I. AND BUCKINGHAM, M. (1988).Transcripts of a^cardiac and (^skeletal aains are early markersfor myogenesis in the mouse embryo. Development 104, 155-164.

SCHNTTZER, J . , FRANKE, W . W . AND SCHACHNER, M. (1981).Immunocytochemical demonstration of vimentin in astrocytesand ependymal cells of developing and adult mouse nervoussystem. /. Cell Biol. 90, 435-447.

SHAW, G., OSBORN, M. AND WEBER, K. (1986). Reactivity of apanel of neurofilament antibodies on phosphorylated anddephosphorylated neurofilaments. Eur. J. Cell Biol. 42, 1-9.

SOLURSH, M. AND MEIER, S. (1986). The distribution of somite-derived myogenic cells during early development in the wingbud. In Somites in Developing Embryos, (ed. R. Bellairs, D. A.Ede and J. W. Lash) Plenum Press, New York, pp.261-275.

TAPSCOTT, S. J., BENNETT, G. S., TOYAMA, Y., KLEINBART, F. AND

HOLTZER, H. (1981). Intermediate filament proteins in thedeveloping chick spinal cord. Devi Biol. 86, 40-54.

THEILER, K. (1972). The House Mouse. Development and NormalStages from Fertilization to 4 Weeks of Age. First edition,Springer-Verlag, Berlin.

TOKUYASU, K. T. AND MAHER, P. A. (1987). Immunocytochemicalstudies of cardiac myofibrillogenesis in early chick embryos. I.Presence of immunofluorescent titin spots in premyofibril stages./. Cell Biol. 105, 2781-2793.

TOKUYASU, K. T., MAHER, P. A. AND SINGER, S. J. (1984).Distributions of vimentin and desmin in developing chickmyotubes in vivo. J. Cell Biol. 98, 1961-1972.

TOWBIN, H., STAEHELIN, T. AND GORDON, J. (1979).Electrophoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets: procedure and some applications. Proc.natn. Acad. Sci. U.S.A. 76, 4350-4354.

TRAUB, P. (1985). Intermediate Filaments. A Review. Springer-Verlag Berlin.

VIEBAHN, C. (1989). Das Muster der Intermediarfilamentproteinewahrend der Histogenese der Somiten beim Kaninchen. Verh.Anat. Ges. 83 (in press).

VIEBAHN, C , LANE, E. B. AND RAMAEKERS, F. C. S. (1987). Themesonephric (Wolffian) and paramesonephric (Mtlllenan) ductsof golden hamsters express different intermediate-filamentproteins during development. Differentiation 34, 175-188.

VIEBAHN, C , LANE, E. B. AND RAMAEKERS, F. C. S. (1988).Keratin and vimentin expression in early organogenesis of therabbit embryo. Cell and Tissue Res. 253, 553-562.

WANG, K., MCCLURE, J. AND TU, A. (1979). Titin: Majormyofibrillar components of striated muscle. Proc. natn. Acad.Sci. U.S.A. 76, 3698-3702.

(Accepted 14 July 1989)

Documents

Desmin and titin expression in early postimplantation ... · Titin was found in the early heart anlage at stage 8.25 d.p.c, when no desmin expression was observed in this tissue