Development 105. 97-107 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

97

Changing patterns of cytokeratins and vimentin in the early chick embryo

MARK PAGE

Department of Anatomy & Developmental Biology, University College London, Gower Street, London, WCJE6BT, UK

Summary

The distribution of cytokeratins and vimentin intermedi-ate filaments in the first 48 h of chick development hasbeen determined using immunofluorescent labelling.During formation of the germ layers, cytokeratin ex-pression is associated with the appearance of an integralepithelium (ectoderm), whereas vimentin expression isassociated with cells that detach and migrate from thisepithelium to form endoderm and mesoderm. Sub-sequently, vimentin persists in the endoderm and meso-derm and the tissues derived therefrom, such as thesomites and developing heart, throughout the period ofstudy. The appearance of cytokeratins at later stages ofdevelopment occurs in some epithelia such as the ecto-derm, endoderm, lateral plate and epimyocardium but

not others including the neural plate, neural tube andsomites. Expression of cytokeratins in endoderm andmesenchymal tissues occurs in tandem with vimentin.

In conclusion, vimentin expression is related to itsdistribution in the epiblast before germ layer formation.Its initial appearance may be related to the motilebehaviour of cells about to ingress through the primitivestreak. The appearance of cytokeratin filaments, how-ever, does not reflect germ layer derivation but ratherthe need for an epithelial sheet.

Key words: chick embryo, cytokeratins, vimentin,intermediate filaments, immunofluorescence.

Introduction

The intermediate filaments are a family of cell-type-specific proteins that appear to be expressed by all cellsso far described (for reviews see Anderton, 1981;Lazarides, 1980, 1982; Osborn & Weber, 1982; Traub,1985). During the first stages of embryogenesis onlyvimentin (mesenchyme-specific) and cytokeratins (epi-thelium-specific) have been observed. These intermedi-ate filaments have been identified even at the oocytestage in Xenopus (Gall et al. 1983; Franz et al. 1983;Godsave et al. 1984a,6; Klymkowsky et al. 1987). Atlater stages of Xenopus development, some of the firstgenes to be expressed that are significantly differentfrom the maternal genome are those encoding for thecytokeratins (Jonas et al. 1985; Winkles et al. 1985;Sargent et al. 1986). In the mouse, cytokeratins andtheir mRNAs have been detected in the oocyte andpreblastocyst stages (Lehtonen etal. 1983; Duprey etal.1985; Lehtonen, 1985) and in stages prior to primitivestreak formation (Jackson etal. 1980, 1981; Paulin et al.1980; Bruletera/. 1980; Kemlerera/. 1981; Oshima etal.1983; Duprey etal. 1985; Chisholm & Houliston, 1987),but vimentin does not appear until the primary mes-enchymal cells have formed (Franke et al. 1982a).Similarly, in quail embryos (Erickson et al. 1987) andchick blastoderm cell cultures (Biehl et al. 1985) onlycytokeratins and vimentin are expressed in the initialstages. Differentiation of muscle, neurones and glial

cells, at later stages of development, is associated withthe first appearance of the other intermediate filaments;these being desmin, neurofilament protein and glialfibrillary acidic protein, respectively (Jackson et al.1981; Raju et al. 1981; Tapscott et al. 1981; Bignami etal. 1982; Franke et al. 1982ft; Tokuyasu et al. 1984;Bovolenta et al. 1984; Erickson et al. 1987). The earlyappearance of the cytokeratins in embryogenesis hasled to the proposal that the formation of epidermis/epithelium is one of the primary events in development,although why this is so is unclear (Sargent et al. 1986).

Despite a relatively comprehensive understanding ofthe biochemistry of the intermediate filaments, theirprecise function and the significance of their cell-typespecificity is still equivocal. The cytokeratins mighthave a special function because of their association withdesmosomes (Franke et al. 1984; Cowin et al. 1985;Jones & Goldman, 1985) in that they maintain theintegrity of epithelial sheets. Experiments that haveattempted to elucidate intermediate filament functionby microinjection of intermediate filament-specific anti-bodies were notable by their failure to disturb normalcell behaviour (Eckert et al. 1981; Gawlitta et al. 1981;Klymkowsky, 1981; Lane & Klymkowsky, 1982; Lin &Feramisco, 1981). This has resulted in a recent challeng-ing theory which proposes that the intermediate fila-ments might act in the regulation of gene expression(Traub, 1985). This idea has arisen from experimentaldata which demonstrate that intermediate filament

98 M. Page

subunit proteins have a high affinity for nucleic acids(Nelson & Traub, 1981; Traub & Nelson, 1982).

As histogenic markers, intermediate filaments canprovide useful information in determining cell differen-tiation events during embryogenesis. The chick embryois particularly valuable in this respect because ourknowledge of cell and tissue movements in its earlydevelopment is more extensive than in any mammalianembryo. Studies on the appearance of intermediatefilaments during development might also provide cluestowards a functional role. Hence, this study describesthe distribution of cytokeratins and vimentin in theearly chick embryo using immunohistochemical me-thodology.

Materials and methods

Preparation of embryos for immunohistochemistryHens' eggs (Ross Brown, Andover, UK) were incubated in arocking incubator at 38°C in humid conditions. The embryoswere then removed from the yolk and vitelline membranes atthe appropriate times to give a range of embryos betweenstages 3 and 12 according to Hamburger & Hamilton (1951).These embryos were placed in Pannett & Compton saline(Pannett & Compton, 1924) and excess yolk removed. Theywere then fixed in either absolute ethanol for 15-30 min or ina solution containing 3-7% paraformaldehyde, 50% ethanoland 4% glacial acetic acid for 45 min. Ethanol-fixed embryoswere rehydrated through a graded series of ethanols (90 %,70 %, 50 %) to PBS, followed by washes with 5 % w/v sucrosein PBS for 2h and finally 15 % sucrose-PBS. Those fixed inparaformaldehyde were washed in PBS and taken to 15 %sucrose-PBS as above. Tissues were stored at 4°C in thissolution to which sodium azide had been added (final concen-tration 0-01%) either overnight or up to 2 weeks. Sub-sequently, the fixed embryos were infiltrated with 7-5%gelatin (300 Bloom; Sigma Chemical Co., Poole, UK) in 15 %sucrose-PBS for 2 h at 37 °C, allowed to set and then frozen inisopentane previously cooled with liquid nitrogen. Transverseserial sections were cut at 8-10 ^m in a Bright cryostat set at—25°C and then mounted on gelatinized slides and stored at4°C until use.

ImmunohistochemistryTissue sections were rinsed in distilled water for 10 min andthen either single- or double-labelled for 15 min to 1 h at 37°Cwith anti-cytokeratin and anti-vimentin antibodies. The anti-bodies were gifts and are detailed in Table 1. In double-labelling experiments, either one of the anti-cytokeratinantibodies was used with the anti-vimentin antibody. Singlelabelled sections were subsequently overlaid with either goatanti-mouse IgG or goat anti-rabbit IgG antibody (as appropri-ate for the primary antibody) conjugated with fluoresceinisothiocyanate (Sigma Chemical Co.) for 30 min at 37°C in thedark. Double-labelled sections were incubated in a cocktailcontaining goat anti-mouse IgG and goat anti-rabbit IgG

antibodies conjugated with rhodamine and fluorescein fluoro-chromes (Sigma), respectively. These second layer antibodieshad previously been passed down Sepharose columns contain-ing bound rabbit or mouse IgGs (as appropriate) to removecross-reacting antibodies. Finally, the sections were washed indistilled water (three changes) and mounted in an aqueousmounting medium (Citifluor, City University, London, UK).They were examined under a Zeiss photomicroscope usingepifluorescence. Photographs were taken on Kodak Tri-X Pan400 and Ektachrome 400 rated at 800 ISO.

In all immunohistochemical procedures, a group of sectionswas included in which either the first, second or bothantibodies were omitted from the protocol. Specific fluor-escence was not observed in these sections.

ImmunoblottingIntermediate-filament-enriched samples were prepared fromembryos at various stages of development; these being 30 h, 3days and 8 days of incubation. For each sample, three dozenembryos were dissected from the yolk and vitelline mem-branes in calcium- and magnesium-free Tyrode's solutioncontaining 5mM-EDTA and OlmM-phenylmethylsulphonylfluoride (PMSF). They were then homogenized by hand witha Douncer (at least 30 strokes) and spun at 11000 g for 10 min.The resultant pellet was resuspended in 0-05M-Tris buffer(pH8-0) containing 1% Nonidet P40 (NP40), 0-15M-NaCland 5mM-EDTA. The sample was respun at 11000 g for10 min and the pellet dissolved in gel loading buffer (0-065 M-Tris (pH6-8) containing 2% sodium dodecyl sulphate, 5% fi-mercapto-ethanol and 10 % glycerol) and heated to 100 °C for2-3 min.

Fractions were also prepared from the skin of prehatchchicks (eggs incubated for 20 days). The skin was cleaned offeathers, fat and muscle in PBS containing 0-1 mM-PMSF, cutinto small pieces and homogenized in a Waring blender. Theskin homogenate was then spun at 15 000 g for 10 min and thepellet resuspended in PBS containing 1 % NP40 and thensonicated, all at 4°C. The suspension was respun and thepellet suspended in gel loading buffer and heated to 100 °C for2-3 min. These samples were stored at —20°C until use.

The skin and embryo samples were run on standardSDS-PAGE (10% acrylamide), transferred to nitrocellulosepaper and labelled overnight at 4°C with the anti-vimentinand anti-cytokeratin antibodies. The labelled proteins weresubsequently visualized by incubation with 125I-donkey anti-rabbit IgG (Sigma) and exposure to X-ray film (Fujii). Arabbit anti-mouse IgG sandwich was included for those blotslabelled with the anti-cytokeratin antibodies.

ImmunoprecipitationNine stage-12 embryos (48 h incubation), dissected free ofyolk and vitelline membranes, were each placed in a well of aMultiwell tissue culture plate (Falcon plastics, type 3008)containing 100^1 of methionine-free Dulbecco's modifiedEagles medium (DMEM). They were incubated in thismedium for 1 h at 37°C (5 % CO2) to deplete endogenousmethionine pools, furthermore, most of the area opaca wastrimmed from these embryos to reduce the methionine input

Table 1. Details of antibodies utilized

Antibody Specificity Reference

LP1K (mouse monoclonal)LP3K (mouse monoclonal)LE65 (mouse monoclonal)Anti-vimentin (rabbit polyclonal)

Recognizes human cytokeratins CK7 (M.54X103) & CK8 (/Wr52-5xl03)Recognizes human cytokeratin CK8 (Mr52-5xl03)Recognizes human cytokeratin CK18 (Mr45xl03)Recognizes chick vimentin ( i )

Lane (personal communication)Lane (personal communication)Lane (1982)Jacobs el at. (1982)

Cytokeratins and vimentin in early chick embryo 99

from yolk granules. The medium was pipetted off andreplaced with 100 (A of labelling medium containing a 1 in 20dilution of L-[35S]methionine (Amersham; SJ 1515,1000Cim-mol"1) in methionine-free DMEM. This gave a final concen-tration of 0-75mCiml~\ The embryos were then incubatedfor a further 4 h at 37 °C.

Preparation of embryo lysates and immunoprecipitationsfollowed the method of Oshima (1981). The medium waswithdrawn and the embryos washed three times in cold PBSfollowed by addition of 1 ml per embryo of SDS harvest buffer(0-1% SDS, lOmM-Tris-HCl (pH7-4), 3mM-MgCl?, 04ITIM-CaC^) containing 0-5 mM-PMSF, 1 mM-N-ethymaleimide and1/100 vol. of aprotinin solution (Sigma), all at 4°C. Theembryos were pooled and dounced in the harvest bufferfollowed by the addition of 45^d DNase I (Wmgrnl"1;Sigma). Further SDS was added to 0-5 % and EDTAadded to1 mM final concentrations. The embryo lysate was then heatedat 100°C for 2min and, after cooling, Nonidet P-40 added to1 %. Aliquots (4 fx\) of the lysate were taken for determiningTCA-precipitable counts and the remainder stored frozen at-20°C. Counts ranged from 5-68xlO4 to 5-73X104 dpm(mean = 5-7xlO4).

Lysates were incubated with the antibodies for 2h at 4°Cfollowed by a rabbit anti-mouse IgG sandwich for lh .Subsequently, formalin-fixed Staphylococcus aureus Cowanstrain I bacteria were added and incubated for 30min. Thebacteria were recovered by centrifugation and washed twicein 0-1% SDS, 1% NP-40, lOmM-Tris-HCl, pH7-4, 5mM-EDTA, twice in 0-5M-NaCl, 50mM-Tris-HCl, pH7-4, 5min-EDTA, 0-05% NP-40 and resuspended in 0-15M-NaCl,50mM-Tris-HCl, pH7-4, 5mM-EDTA, 0-05% NP-40. Theradioactive proteins and immunoglobulins were solubilized ingel loading buffer, heated to 100 °C for 2 min and separated onpolyacrylamide gels. The gels were processed for fluorogra-phy with 2,5-diphenyloxazole-dimethyl sulphoxide and ex-posed to Kodak X-ray film at -70°C.

Results

The patterns of immunofluorescence following treat-ment with LP1K, LP3K and LE65 antibodies wereidentical in all stages examined and are describedtogether. Fixation in absolute ethanol gave optimalimmunofluorescence for the anti-cytokeratin antibodiesbut morphological preservation was poor; the use ofother fixatives drastically reduced or completely abol-ished antigenicity. The anti-vimentin antibody was lesssusceptible to the fixative used, but those embryos fixedin paraformaldehyde gave superior immunofluor-escence and morphological preservation.

Analysis of antibody specificityThe specificity of the anti-intermediate filament anti-bodies was analysed by immunoblotting and immuno-precipitation. LP3K and LE65 antibodies did not reactwith blotted proteins or precipitate protein from radio-labelled embryos (data not shown); LP1K, however,was found to recognize a single band of an apparentmolecular weight of 56000 in intermediate filament-enriched material prepared from embryos incubated for30h, 3 days (not shown) and 8 days (Fig. 1). Thisprotein band was not resolved in skin fractions (Fig. 1).

The anti-vimentin antibody recognizes a major bandof apparent molecular weight 58 000 in immunoblots ofembryonic material (30 h, 3 days (not shown) and 8days) and chick skin (Fig. 1). Immunoprecipitation dataconfirmed this finding in stage-12 embryos (Fig. 1).

Stages 3-4 (12-19 h)The primitive streak is the first readily observablestructure to form at around 6h of incubation and by

I•+V

— V

8

Fig. 1. Immunoblotting and immunoprecipitation data of chick material probed with LP1K (lanes 1-3) and anti-vimentin(lanes 4-8) antibodies. Lanes 1-3 and 6-7 are taken from the same gel. LP1K does not recognize NP40-insoluble proteins inchick skin (lane 1) but does recognize a protein band of apparent molecular weight 56000 (CK) in both 30 h (lane 2) and 8day (lane 3) embryos. The anti-vimentin antibody recognizes a protein band of apparent molecular weight 58000 (V) in 30 h(lane 4) and, in another gel, 8-day embryos (lane 5). NP40-insoluble fractions of chick skin (lane 6) and a cell lysateprepared from chick skin fibroblasts (lane 7) also demonstrate the 58000 band. The antibody also precipitates a 58000Mrprotein from 48 h embryos (lane 8). The lower bands of apparent molecular weight 45000 (arrow) in lanes 4-7 are probablybreakdown products resulting from the enrichment procedure. The high molecular weight bands in lane 4 were found to benonspecific when compared with blots from the same gel that had not been incubated with specific antibody.

100 M Page

A

f

Fig. 2. Diagrammatic representations of a stage-4 embryoshowing dorsal view (A) and cross section (B).B corresponds to line X in A. The arrows (B) indicatemigratory routes of the cells that leave the ectoderm. Theboxed areas marked 3 and 4 represent comparative areas inFigs 3 and 4, respectively, ao, area opaca; ap, areapellucida; Hn, Hensen's node; ps, primitive streak;ec, ectoderm; m, mesoderm; en, endoderm; ye, yolkyendoderm of area opaca.

about 19 h extends two thirds of the way across the areapellucida along the craniocaudal axis (Hamilton, 1952)(Fig. 2A). At this stage the embryo consists of threegerm layers; the ectoderm, mesoderm and endoderm(Fig. 2B).

Anti-cytokeratins (Table 2)LP1K/LP3K/LE65 antibody binding at these stages,was restricted mostly to the ectoderm cells at theperimeter of the embryonic disc overlying the areaopaca and edge of the area pellucida (marginal zone)(Fig. 3). In these early stages, the apical regions of thecells were preferentially labelled (Fig. 3). A few cells inthe mesoderm and endoderm were also labelled (Figs 3,4); those in the endoderm were restricted to peripheralregions (Fig. 3).

Anti-vimentin (Table 3)Anti-vimentin antibody labelling of the ectoderm wasobserved in central regions of the embryonic disc,including the primitive streak (Fig. 4), but only oc-casional cells were labelled in the peripheral ectodermoverlying the area opaca and marginal zone (Fig. 3).The mesoderm and endoderm showed moderate fluor-escence (Figs 3, 4).

Table

Tissue

2. LP1K/LP3K/LE65 labelling of chickStage

3-4 5-7

tissues

8-12

ap

Hn

ps

€C

m

ye

EctodermEndodermMesodermNeural plate/tubePrimitive streakPrecardiac mesodermPre-foregut/foregutNotochordSegmental plateLateral plate

-somatic-splanchnic

SomitesHeart

-endocardium-epimyocardium

0 = tissue not present+ = weak fluorescence+ + = strong fluorescence- = no fluorescence* strong fluorescence in area

pellucidat scattered cells only

+*-+t0—0000

000

00

opaca

+ +++t

++ ++—0

000

00

and marginal zone of area

+++++t+

+ +

Table 3. Anti-vimentin labelling of chick tissues

Tissue

EctodermEndodermMesodermNeural plate/tubePrimitive streakPrecardiac mesodermPre-foregut/foregutNotochordSegmental plateLateral plate

-somatic-splanchnic

SomitesHeart

-endocardium-epimyocardium

0 = tissue not present4- = weak fluorescence4-4- = strong fluorescence— = no fluorescence+/- = variable fluorescence* area pellucida onlyt medial and caudal regions

3-4

4-*++04-0000

000

00

onlyX fluorescence most prominent in basal

Stage

5-7 8-12

+t +/-

+/- +/-+t +

+/- +/—4-4- +4-4- 4-

+/— + /~0 +X

0 +t0 ++X0 +t

0 4-0 4-4-

and lateral regions

Double-labelling experiments revealed that anti-cytokeratin and anti-vimentin antibody distributionswere largely mutually exclusive except for occasionalcells in the ectoderm and endoderm (Figs 3, 4).

Stages 5-7 (19-26h)The embryo at this stage possesses a head process,

Fig. 3. Transverse section through the marginal zone andarea opaca of a stage-4 embryo, double labelled with LE65and anti-vimentin antibodies. Compare with boxed area 3 inFig. 2B. Cytokeratin (red) is located mostly in the apicalregions of the ectoderm cells (ec). Vimentin (green) occursin the mesoderm (m) and endoderm (en), but onlyoccasionally in the ectoderm where it is coexpressed (yellowfluorescence; arrowhead). A few cells in the mesoderm andyolky endoderm are double labelled (arrows). Ethanol-fixed. Scale bar, 50 nm.Fig. 4. Transverse section through the primitive streakregion of a stage-4 embryo double labelled with LE65 andanti-vimentin antibodies. Compare with boxed area 4 inFig. 2B. Vimentin is located in the ectoderm (ec),mesoderm (m) and endoderm {en), but cytokeratin occursin only a few cells which are double labelled (arrowheads).ps, primitive streak. Scale bar, 50 fim.Fig. 6. Transverse section through the neural plate regionof stage-6 embryo double labelled with LE65 and anti-vimentin antibodies. Compare with boxed area 6 in Fig. 5B.Little red or yellow fluorescence, indicative of cytokeratin,is apparent in any of the cell layers. Vimentin, however(green fluorescence), is located in the neural plate cells(np), endoderm (en) and mesoderm (m). Ethanol-fixed.Scale bar, 50 fim.Fig. 7. Transverse section through the area pellucida andlateral margin of the neural plate of a stage-6 embryodouble labelled with LE65 and anti-vimentin antibodies.Compare with boxed area 7 in Fig. 5B. Cytokeratin (red) islocated in the ectoderm (ec) up to the lateral margin(arrowhead) of the neural plate (np). The endoderm (en)shows only limited red fluorescence. Vimentin is located inthe neural plate, endoderm and mesoderm (in), but showsonly limited distribution in the ectoderm adjacent to theneural plate where it overlaps with cytokeratin distribution.Some cells in the ectoderm are double labelled (arrow).Ethanol-fixed. Scale bar, 50fim.Fig. 8. Transverse section through the centripetal region ofthe area pellucida of a stage-6 embryo double labelled withLE65 and anti-vimentin antibodies. Compare with boxedarea 8 in Fig. 5C. The primitive streak lies just outside theright edge of the frame. Low levels of red fluorescence,indicating cytokeratin, are present in the ectoderm (ec) andendoderm (en) nearly up to the midline (primitive streak).Vimentin is located in the mesoderm (m) and endoderm,but shows limited fluorescence in the ectoderm where itoverlaps with cytokeratin distribution. Some cell in theectoderm are double labelled (arrow). Ethanol-fixed. Scalebar, 50/im.

Fig. 13. Transverse section through the region of the cardiac fold in a stage-8 embryo double labelled with LE65 and anti-vimentin antibodies. Compare with boxed area in Fig. IOC. Many cells in the endoderm (en) and splanchnic mesoderm(spin) are double labelled and hence fluoresce yellow, although individual red and green filaments can also be seen in theselayers. Cytokeratin and vimentin also occur in the somatic mesoderm (som) and ectoderm (ec). Ethanol-fixed. Scale bar,50;imi.Fig. 14. Transverse section through the heart region of a stage-12 embryo double labelled with LE65 and anti-vimentinantibodies. Compare with the boxed area in Fig. 10B. Cytokeratin is located in the ectoderm (ec), endodermal lining of theforegut (fg) and epimyocardium (epc). In all of these layers, some cells are double labelled (yellow fluorescence). Theneural tube (nt) and endocardium (enc) are unreactive. Vimentin is located in all tissues, although the ectoderm (ec) showsonly scattered fluorescence, m, mesoderm. Ethanol-fixed. Scale bar, 50,«m.

Cytokeratins and vimentln in early chick embryo 101

X-

Y-

ps

Fig. 5. Diagrammatic representations of a stage-6 embryoshowing (A) dorsal view, (B) cross section through theneural plate region and (C) cross section through primitivestreak region. B and C correspond to lines X and Y in A,respectively. The boxed areas numbered 6, 7 and 8represent comparative areas in Figs 6, 7 and 8, respectively.The arrows (C) indicate migratory routes of cells as theyleave the ectoderm, ao, area opaca; ap, area pellucida;no, notochord; Hn, Hensen's node; ps, primitive streak;np, neural plate; m, mesoderm; en, endoderm;ec, ectoderm; ye, yolky ectoderm of area opaca.

Hensen's node and a definitive primitive streak, but isstill composed three germ layers (Fig. 5). The neuralplate develops following the craniocaudal regression ofHensen's node and subsequent notochord formation. Itcan be identified as a layer of columnar cells in theectoderm which is henceforth distinguished as eitherneural or non-neural (Fig. 5B). The head fold begins toform at about stage 6 and the first pair of somitesdevelop at stage 7.

Anti-cytokeratins (Table 2)At these stages, the distribution of immunofluorescentcells in the ectoderm and endoderm is more extensivethan in younger embryos. Immunoreactivity persists inperipheral regions but it has also progressed centrip-etally. In rostral regions, the non-neural ectoderm islabelled entirely, but the neural ectoderm (neural plate)is unreactive apart from the cells at its lateral margin(Figs 6, 7). Similarly, fluorescent cells in the endodermare present both peripherally and centripetally, butonly a few cells are labelled in the midline (Figs 6, 7). In

caudal regions, the ectoderm and endoderm exhibitimmunoreactivity peripherally and centripetally but notin the midline (Fig. 8).

Anti-vimentin (Table 3)Anti-vimentin antibody labelling of the neural and non-neural ectoderm exhibited a reversed staining patternto that for the anti-cytokeratin antibodies. Hence, theneural plate is immunostained throughout (Fig. 6), butthe non-neural ectoderm is generally unreactive apartfrom a few isolated cells (Fig. 7). In caudal regions,binding of the anti-vimentin antibody to the ectoderm,at these stages, becomes more restricted to centripetalregions of the embryonic disc and only limited fluor-escence is seen (Fig. 8). The endoderm and mesodermhowever, are labelled in all areas of the blastoderm(Figs 6, 7, 8).

Double-labelled sections showed areas of fluorescentoverlap in the ectoderm, the endoderm and at thelateral margins of the neural plate (Figs 7, 8), but, as inearlier stages, the immunostaining patterns showed anoverall mutual exclusiveness.

Stages 8-12 (24-49 h)During these stages Hensen's node continues to regressand to form notochord (Fig. 9). The head fold becomesmore pronounced and the heart primordia develop tofuse eventually in the midline, forming the heart(Fig. 10). The folding of the endoderm during heartdevelopment results in the formation of the foregut(Fig. 10B,C). The somites continue to form in a cranio-caudal sequence from the segmental plates situatedeither side of the neural tube (Fig. 9).

Anti-cytokeratins (Table 2)Caudally, the primitive streak persists and the patternof immunoreactivity in the ectoderm and endoderm isidentical to that seen at earlier stages. In more cranialsections, however, both the ectoderm and endodermare fluorescent in all regions (Figs 11A, 12A, 13, 14).The fluorescent ectoderm can be distinguished clearlyfrom the unlabelled neural tube (cf. unlabelled neuralplate, Fig. 6), but where it has not closed dorsally, somecells (neural crest/tube?) lying on the medial aspectwere also labelled (Fig. 12A). Once the tube has closed,as seen in more cranial sections and at later stages, onlythe ectoderm is fluorescent (see Fig. 11 A). In the regionof the heart folds, the endoderm and associatedsplanchnic mesoderm are strongly labelled (Fig. 13).The structures that are derived from these layers (theforegut and heart, respectively) also exhibit immuno-reactivity (Fig. 14). The precardiac (splanchnic) meso-derm develops into the epimyocardium and endocar-dium, but only the former is immunoreactive (Fig. 14).The somatic mesoderm is also fluorescently labelled(Fig. 13).

The notochord, neural tube, somites and segmentalplate are unreactive (Figs 11A, 12A, 14), but thesomatic and splanchnic components of the lateral plate(Fig. 11A) and the extraembryonic membranes (notshown) are strongly fluorescent.

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Fig. 9. Diagrammatic representation of the caudal region ofa stage-10 to -12 embryo showing (A) dorsal view, (B) crosssection through the somites and (C) cross section throughthe segmental plate. B and C correspond to lines X and Yin A, respectively. Compare B and C with Figs 11 and 12,respectively, s, somite; nt, neural tube; sp, segmental plate;Hn, Hensen's node; ps, primitive streak; Ip, lateral plate.

Anti-vimentin (Table 3)Anti-vimentin antibody labelling at these stages is moreextensive and is seen in most tissues that have differen-tiated, such as the somites, segmental plate, lateralplate and neural tube (Figs 11B, 12B). In these epi-thelial tissues, the fluorescence showed a marked baso-lateral distribution. The ectoderm is weakly labelledbut the endoderm shows strong fluorescence in all areasof the embryonic disc (Figs 11B, 12B, 13, 14). Thesplanchnic and somatic mesoderm in the heart foldregion also exhibit fluorescence (Fig. 13) and as theheart develops both the epimyocardium and endocar-dium are labelled (Fig. 14). In the lateral plate, thesplanchnic mesoderm is more strongly labelled than thesomatic component (Fig. 11B).

Double-labelled sections revealed large regions offluorescent overlap in the splanchnic and somatic meso-derm, epimyocardium, foregut, endoderm (Figs 13,14)

ap

Fig. 10. Diagrammatic representation of the cranial regionof an idealized, stage-10 to -12 embryo showing (A) dorsalview, (B) cross section through heart region and (C) crosssection through heart folds before heart formation. Theheart in A is drawn as it appears following fusion of theheart tubes (see C). B and C correspond broadly to lines Xand Y in A, but are at different stages of development. Theboxed areas numbered 13 and 14 represent comparativeareas in Figs 13 and 14, respectively. Note, however, thatthe heart tubes in C have not formed in Fig. 13 and that theposition of the heart in Fig. 14 has been displaced to theright of the neural tube and pharynx, ap, area pellucida;ov, optic vesicle; h, heart; hb, hindbrain; 5, somite;ph, pharynx; spm, splanchnic mesoderm; ht, heart tube.

and lateral plate (not shown, but compare Fig. 11A and11B).

Discussion

The main findings of this paper demonstrate thatcytokeratins and vimentin intermediate filaments areexpressed at early stages of chick embryogenesis. Theanti-cytokeratin antibodies utilized in this study recog-nize the human cytokeratins designated CK7, CK8 andCK18 (Moll et al. 1982). These polypeptides are charac-

Cytokeratins and vimentin in early chick embryo 103

Fig. 11. Transverse sections through the somite region of stage-11 (A) and stage-10 (B) embryos immunofluorescentlylabelled with LP3K (A) and anti-vimentin (B) antibodies. Compare these sections with the diagrams in Fig. 9B.(A) Cytokeratin is located in the ectoderm (ec), endoderm (en) and lateral plate mesoderm (Ip). Note apical distribution inlateral plate cells. The somites (s), neural tube (nt) and notochord (no) are unreactive. (B) Vimentin is located in thebasolateral regions of the neural tube cells (nt), somites (s), and lateral plate (lp). Note the splanchnic mesoderm is morefluorescent than the somatic and weak fluorescence in the endoderm (en) and notochord (no). The ectoderm (ec) is mostlyunlabelled. (A) Ethanol-fixed, (B) paraformaldehyde-fixed. Scale bar, 50/an.

teristically expressed in embryonic and simple epitheliain which the CK8 and CK18 proteins interact specifi-cally to form 8nm keratin filaments (Fuchs et al. 1984);CK7 can also interact with CK18 (Sun et al. 1984). Thecodistribution of these cytokeratins in chick embryonicepithelia concurs with these findings. The results indi-cate that the LP1K antibody recognizes a filamentous,intracellular protein in embryonic tissues of apparentmolecular weight 56000 which is not expressed in thecomplex epithelium of the chick skin. Such data providepersuasive evidence that the LP1K, LP3K and LE65antibodies specifically recognize a cytokeratin filamentin chick embryos.

The anti-vimentin antibody recognizes a 58 000 mol-ecular weight protein in chick tissues as shown by

immunoblotting and immunoprecipitation (Fig. 1); thisagrees with previous findings using this antibody(Jacobs etal. 1982).

The initial patterns of expression of cytokeratins andvimentin in the primitive streak stages of the chickembryo can be related to the cell migrations that occurat this time (see Bellairs, 1986). There appears to beonly limited coexpression of vimentin and cytokeratins.The cytokeratins are restricted, at first, to the ectodermoverlying the area opaca and marginal zone and areassociated with cells destined to remain in the ecto-derm, whereas vimentin is localized to centripetalregions of the ectoderm and is associated with cellsdestined to ingress through the streak to form primarymesoderm and definitive endoderm. The subsequent

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Fig. 12. Transverse sections through the segmental plate region of a stage-11 (A) and stage-8 (B) embryoimmunofluorescently labelled with LP3K (A) and anti-vimentin (B) antibodies. Compare these sections with the diagrams inFig. 9C. (A) Cytokeratin is located in the gap (arrow) between the two apposing crests of the neural tube which has justclosed. Note that the ectoderm (ec) and endoderm (en) are strongly fluorescent. (B) Vimentin is located in the basolateraregions of the cells of the neural tube (nt), segmental plate (sp) and notochord (no). The endoderm (en) and some cells inthe ectoderm (ec) are also fluorescent. (A) ethanol-fixed (B) paraformaldehyde-fixed. Scale bar, 50 fim.

expression of vimentin in mesodermal and endodermaltissues may therefore be attributed to these migratorycells.

The distribution of cytokeratins at the primitivestreak stage correlates with that of desmosomes whichare restricted to the peripheral ectoderm (area opaca)and marginal zone of the area pellucida (Overton, 1962;Bellairs, 1986; Bellairs et al. 1978). It is likely that thedesmosomes and cytokeratin filaments are closely as-sociated in this ectoderm for the following reasons: (a)the cytokeratins and desmosomes are apically distrib-uted, (b) the appearance of desmosomal proteins(desmoplakins) and cytokeratin filaments are corre-lated during development (Jackson et al. 1980) and (c)the desmosome-tonofilament (cytokeratin) complex ischaracteristic of epithelia (Franke et al. 1978, 1984;Jones & Goldman, 1985). The function of this complexis to maintain the integrity of the epithelial sheet and

must be a vital requirement for the chick blastodermundergoing centrifugal tension during the primitivestreak stage (Bellairs et al. 1967).

The initial absence of cytokeratins in the chickendoderm is considered unusual because this epi-thelium, along with the ectoderm, is one of the first celllayers to be formed. However, the endoderm mayexpress other cytokeratin filaments, characteristic ofsimple epithelia (e.g. CK19 in humans; Sun et al. 1984)which are not recognized by the antibodies used in thepresent study. The endoderm is initially squamousrather than cuboidal (as in the ectoderm) and only lateris a cuboidal morphology assumed, by which timeimmunoreactivity is apparent. Hence, epithelial cellmorphology may reflect the type of intermediate fila-ment expressed (see Connell & Rheinwald, 1983; BenZe'ev, 1984) and also the cytokeratin composition (seeQuinlane/a/. 1985).

Cytokeratins and vimentin in early chick embryo 11)5

The appearance of vimentin in the endoderm andmesoderm may be related to the motile behaviour ofthese cells as they detach from the ectoderm duringingression. Other studies have also associated vimentinexpression with reduced cell-cell contact, cell spread-ing and growth (Connell & Rheinwald, 1983; BenZe'ev, 1984). These authors have demonstrated thatepithelial cells switch their normal cytokeratin syntheticpattern to a predominantly vimentin profile in low celldensities and vice versa at high cell densities.

In the mouse embryo, the first appearance of vimen-tin is seen in mesenchymal cells only (Franke et al.1982a). Cytokeratins are expressed across the entireectoderm and the desmosome-cytokeratin complex islost as cells leave the primitive streak. Variations iningression events between mammals and birds (seePoelmann, 1981) might account for these differences.

Differentiation of the ectoderm into neural and non-neural components shows a marked correlation with theappearance of vimentin and cytokeratins, respectively.There are only small regions of coexpression in thelateral margins of the neural plate. The absence ofcytokeratins in presumptive neural tissue is consistentwith other studies (Tapscott et al. 1981; Erickson et al.1987) but is perhaps surprising in an epithelium thatundergoes severe shape changes and hence wouldrequire structural unity. This may however, be providedby the basolaterally distributed vimentin filaments as-sociating with hemidesmosomes (see Kartenbeck et al.1984).

Closure of the neural tube is associated with theappearance of cytokeratins in the neural crest andmight be a preparatory event for ectodermal sealing byformation of desmosome-tonofilament complexes.

Subsequent expression of cytokeratins in regionsother than the ectoderm is complementary to vimentinexpression, for example, in the endoderm, lateral plateand precardiac mesoderm (later to become the epimyo-cardium). In these regions, the appearance of cytokera-tins might be indicative of an 'epithelialization' event.In the heart folds, for example, both the mesoderm andendoderm experience tensions during the folding pro-cess and hence there is a requirement for an integralsheet to withstand these forces.

The early differentiation events in the chick embryoare not associated with transitions of expression fromone intermediate filament type to another, whereas atlater stages of development, for example, there is anapparent replacement of vimentin by desmin duringmyogenesis (Bennett et al. 1979; Holtzer et al. 1981) orby neurofilament protein during neurogenesis (Tapscottet al. 1981). Vimentin is not replaced by cytokeratinsduring formation of the foregut and heart for example,and transitions of intermediate filament expressionappear to be related therefore to terminal differen-tiations of functional adult tissues such as nerve andmuscle. Coexpression of cytokeratins and vimentin maybe an important feature of the relatively undifferen-tiated tissues of the embryo and appears to be awidespread trait that has not been fully recognized (seeLane et al. 1983).

As cell-type-specific markers, the distribution ofintermediate filaments should reflect histogenic lineage.The expression of cytokeratin and vimentin filamentsduring chick embryogenesis, however, appears to berelated more to functional and behavioural require-ments rather than to germ layer derivation (see Erick-son et al. 1987).

This work was funded by Action Research for the CrippledChild (Grant No. A/8/1526) and The British Heart Foun-dation (Grant No. 86/88). I am grateful to Drs B. Lane and P.Hollenbeck for providing antibodies, to Ros Cleevely and LizHarfst for expert technical assistance and to Professor RuthBellairs for reading the manuscript.

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{Accepted 8 October 1988)