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We disrupted mouse Crkas by inserting a neomycin-resistance cassette into an exon encoding a part of the SH3 domain (Fig. 1a ). Correctly targeted embryonic stem (ES) cells were used to create chimaeric males, which transmitted the mutated allele through the germ line. Mice heterozygous for Crkas showed no phenotypic changes and were intercrossed to generate homozygous mutants. Southern- and western-blot analysis of the offspring showed that the gene targeting resulted in a null mutation of Crkas (Fig. 1b,c).

Figure 1: Targeted disruption of mouse Crkas.
figure 1

a, Targeting strategy. The disrupted exon (Exon), neomycin-resistance gene (Neo) and diphtheria toxin-A gene (DTA) are shown as black, white and shaded boxes, respectively. Restriction enzymes are: B, BamHI; H, HindIII; X, XhoI; S, SalI; and C, ClaI. The expected lengths of BamHI-digested genomic fragments are also shown. b, Southern blot for genotyping embryos. BamHI-digested DNA samples from wild-type (+/+), heterozygous (+/–) and homozygous (–/–) embryos were hybridized with 5´ and 3´ probes shown in (a) . G, germline band; T, targeted band. c, Western blot of a homozygous embryo showing absence of Cas. The position of Cas is indicated by an arrow.

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Cas-deficient embryos, obtained by caesarian section, appeared phenotypically abnormal at 11.5–12.5 days post coitum (dpc), showing systemic congestion and growth retardation (Fig. 2a). Histological examination revealed consistent abnormalities in the heart and blood vessels (Fig. 2b). In comparison with wild-type littermates (Fig. 2b, right), Cas-deficient embryos showed a poorly developed heart, consisting of thin myocardium, and prominently dilated blood vessels that retained blood cells (Fig. 2b, left). The liver was occasionally smaller in size, but no obvious differences were observed in other organs, including the central nervous system, lungs, kidneys and skeletal muscles, at this stage (data not shown). Normal embryos stained with anti-Cas antibody1 showed that at 11.5–12.5 dpc, when Cas-deficient embryos exhibited the abnormal phenotype, Cas was preferentially expressed in heart muscles (Fig. 2c) and in blood vessels (data not shown). The coincidence of Cas-immunoreactivity and histological abnormalities suggests that lack of Cas impaired cardiac development and blood vessel integrity. Retarded liver growth was considered to be secondary effect, as Cas protein was not detected in hepatocytes (data not shown).

Figure 2: Histopathological analyses of embryos.
figure 2

a, Macroscopic appearances of Cas-deficient (–/–) and wild-type (+/+) embryos at 12.5 dpc. The Cas-deficient embryo shows marked systemic congestion and growth retardation. b, Sagittal H&E-stained sections of Cas-deficient (–/–) and wild-type (+/+) embryos at 12.5 dpc. The boxed areas are magnified in the insets below. In the Cas-deficient embryo, the poorly developed heart is indicated by an arrow and dilated superior vena cava and dorsal aorta are indicated by solid and open triangles, respectively. c, Immunohistochemical staining of a wild-type embryo at 11.5 dpc. The boxed area is magnified in the right inset. The positive signals are preferentially detected in the heart muscles. d, Electron microscopic analyses of Cas-deficient (–/–) and wild-type (+/+) hearts at 12.5 dpc. The boxed areas in the upper panels are magnified in the lower panels. Note the disorganized myofibrils and disrupted Z-disks in the Cas-deficient cardiocyte. Intact Z-disks in the wild-type cardiocyte and a disrupted Z-disk in the Cas-deficient cardiocyte are indicated by solid triangles. e, Immunofluorescent staining of a normal heart with anti-Cas and anti-actinin antibodies. Localization of Cas and actinin are visualized by red and green signals (left and middle), respectively, and co-localization of Cas and actinin is seen as yellow-green signals (right).

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Electron microscopic analyses of the heart revealed alterations in contractile structures of Cas-deficient cardiocytes. Wild-type cardiocytes showed orderly arranged myofibrils and regularly repeated Z-disks (Fig. 2 d, right), whereas Cas-deficient cardiocytes exhibited disorganized myofibrils and disrupted Z-disks (Fig. 2d, left). The intracellular localization of Cas was examined by staining wild-type cardiocytes with anti-Cas antibody1 and staining with anti-actinin antibody was done to identify Z-disks14. Cas was co-localized with actinin (Fig. 2e), indicating that Cas is mainly localized at Z-disks in cardiocytes and is essential in organization of myofibrils and in maintaining integrity of Z-disks.

These data demonstrate that Cas is essential for embryogenesis, particularly in cardiovascular development. Although several Cas-related molecules, Efs/Sin (refs 15,16) and HEF1/Cas-L (refs 17,18), have been identified, the embryonic lethality of Cas-deficient mice indicates that Cas is important in embryogenesis and that other Cas-related molecules cannot fully compensate for Cas. Impaired heart development has been reported in mice lacking several proteins, such as transcriptional enhancer factor-1 (ref. 19), gp130 (ref. 20), or GATA-4 (ref. 21). Unlike the case of Cas-deficient mice, mice lacking these genes showed normal myofibril organization and intact Z-disks19,20,21. Therefore, Cas-deficient mice appear to present a case in which the disorganization of subcellular structures underlie cardiovascular defects. The finding that Cas-deficient embryos die at 11.5–12.5 dpc, a period when the heart begins productive beating22, suggests that the disruption of contractile structures results in cardiac pump failure and venous dilatation, which leads to low blood pressure, systemic hypoxia and embryonic death.

To investigate the role of Cas in the cytoskeletal framework, primary fibroblasts were established from wild-type and Cas-deficient embryos. Morphologically, Cas-deficient fibroblasts were flat, thin and round-shaped in comparison with wild-type cells (Figs 3a,b and 4a,b). Staining with phalloidin revealed changes in distribution and organization of the actin cytoskeleton. Whereas wild-type fibroblasts featured thick, dense and long actin bundles traversing the cells, Cas-deficient fibroblasts exhibited thin, short and irregularly assembled actin filaments at the cell periphery (Fig. 3a,b). In contrast, focal adhesion proteins, such as vinculin, Fak, talin and tensin, accumulated at the ends of the actin filaments in both types of cells ( Fig. 3c,d, and data not shown). Transient re-expression of Cas in Cas-deficient cells displayed restoration of actin stress fiber formation (Fig. 3e–j), demonstrating that the impaired actin assembly was due to Cas-deficiency. These results indicate that Cas is essential in assembling actin filaments and in forming long actin fibers. Similar cytoskeletal changes were noted in Fak-deficient cells, where focal adhesion formation was present but long actin fibers were absent23. These coincident findings suggest that Cas and Fak may coordinately function at a critical step for actin filaments to assemble and grow from focal adhesions.

Figure 3: Immunofluorescent analyses of primary fibroblasts.
figure 3

Staining of wild-type (+/+, a,c) and Cas-deficient (–/–, b,d) fibroblasts with phalloidin (a,b) and anti-vinculin antibody (c,d). Note the impaired actin stress fiber formation in the Cas-deficient cell. Staining of Cas-deficient (–/–, e,h) and Cas-re-expressing (–/– and Cas, f, g,i,j) fibroblasts with anti-Cas antibody (e– g) and phalloidin (h–j). The middle panels (f, i) show a representative cell that re-expressed Cas and demonstrated restoration of stress fiber formation. In the right panels (g,j), among three cells shown, only the upper right one that re-expressed Cas demonstrated restoration of stress fiber formation.

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Figure 4: Morphological, biochemical and immunofluorescent analyses of primary fibroblasts expressing a-Src.
figure 4

Representative morphological appearances of wild-type (+/+, a), Cas-deficient (–/–, b), a-Src-expressing, wild-type (+/+ and a-Src, c), a-Src-expressing, Cas-deficient (–/– and a-Src, d) and a-Src-expressing, Cas-re-expressing cells (–/– and a-Src and Cas, e). Note that a-Src expression in wild-type cells induces a typical transformed morphology (c), whereas a-Src expression in Cas-deficient cells induces only elongated and spindle-shaped features (d), and re-expression of Cas converts this to the transformed appearance (e). f, Expression of Cas and a-Src in the cells shown in (a–e). Similar amounts of protein aliquots were blotted with anti-Cas or anti-Myc antibody. The positions of Cas and a-Src are indicated by arrows. g, Tyrosine-phosphorylation of Cas in the cells shown in (a–e). Similar amounts of protein aliquots were immunoprecipitated with anti-Cas antibody and immunoprecipitants were blotted with anti-phosphotyrosine antibody. Increased tyrosine-phosphorylation of the endogenous and re-expressed Cas is observed in a-Src-expressing, wild-type (+/+ and a-Src) and a-Src-expressing, Cas-re-expressing (–/– and a-Src and Cas) cells (indicated by P-Cas). The positions of immunoglobulin (Ig) and co-immunoprecipitated Src (Src; ref. 1) are also indicated. h, Tyrosine-phosphorylation of cellular proteins in the cells shown in (a –e). Similar amounts of protein aliquots were blotted with anti-phosphotyrosine antibody. a-Src-expressing, wild-type (+/+ and a-Src) and a-Src-expressing, Cas-deficient (–/– and a-Src) cells show similar tyrosine-phosphorylation patterns, and re-expression of Cas into a-Src-expressing, Cas-deficient cells has no substantial change in the tyrosine-phosphorylation pattern (–/– and a-Src and Cas). Immunofluorescent staining of a-Src-expressing, wild-type (+/+ and a-Src, i,k,m) and a-Src-expressing, Cas-deficient (–/– and a-Src, j, l,n) cells with phalloidin (i,j), anti-Fak ( k,l) and anti-Src (m,n) antibodies. Actin filaments, Fak and Src are visualized by red, green and blue signals, respectively. In a-Src-expressing, wild-type cells, actin filaments, Fak and Src are all localized to podosomes (i,k,m). In contrast, in a-Src-expressing, Cas-deficient cells, accumulation of actin filaments in podosomes is incomplete and residual actin filaments are observed in the cytoplasm (indicated by arrows in j), whereas Fak and Src are almost completely localized to podosomes (l,n).

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To examine the roles of Cas in Src-induced transformation, constitutively activated Src with Myc-tag (termed a-Src) was introduced into primary fibroblasts and stable transformants were established. Transfection of a-Src into wild-type and Cas-deficient cells produced a number of colonies with different morphological appearances. The expression of a-Src in these colonies was examined by anti-Myc immunoblotting, and several independent wild-type and Cas-deficient colonies expressing similar amounts of a-Src were selected and analysed. Morphologically, wild-type colonies expressing a-Src showed a transformed phenotype (rounded and refractile), whereas none of Cas-deficient colonies expressing comparable amounts of a-Src displayed a transformed phenotype; they showed an elongated and spindle-shaped appearance. To investigate whether this morphological disparity was due to Cas-deficiency, Cas was introduced into a-Src-expressing, Cas-deficient cells. Cas transfection resulted in appearance of colonies displaying a rounded and refractile morphology (Fig. 4a–3) and immunoblot analyses revealed that morphologically transformed colonies re-expressed Cas in addition to a-Src (Fig. 4f). The mobility shift of the endogenous and re-expressed Cas in a-Src-expressing cells (Fig. 4f) was attributed to increased tyrosine-phosphorylation1, which was demonstrated by blotting anti-Cas immunoprecipitants with anti-phosphotyrosine antibody (Fig. 4g). The transforming activity of a-Src in these cells was further analysed by anchorage-independent cell growth. Repeated experiments using independent clones expressing similar amounts of a-Src had the same results. a-Src-expressing, wild-type and a-Src-expressing, Cas-re-expressing cells formed a significant number of colonies, whereas a-Src-expressing, Cas-deficient as well as wild-type and Cas-deficient cells did not develop any colonies (Table 1). These results have demonstrated that Cas is required for a-Src induction of morphological transformation and colony formation in soft agar. To explain the loss of transforming ability of a-Src in Cas-deficient cells, we examined tyrosine-phosphorylation of cellular proteins and re-organization of cytoskeletal and focal adhesion proteins, which have been considered necessary for Src-induced transforming processes24,25,26. Tyrosine-phosphorylation was similarly induced in a-Src-expressing, wild-type and a-Src-expressing, Cas-deficient cells (Fig. 4h), and re-expression of Cas in a-Src-expressing, Cas-deficient cells did not substantially affect the tyrosine-phosphorylation pattern, indicating that the phosphotyrosyl activity of a-Src was not impaired by Cas-deficiency. On the other hand, immunofluorescent staining showed a significant difference in the re-distribution patterns of actin cytoskeleton. In a-Src-expressing, wild-type cells, actin filaments, Fak and Src were highly concentrated in podosomes, a focal adhesion structure observed in transformed cells27 (Fig. 4i,k,m ). In contrast, in a-Src-expressing, Cas-deficient cells, accumulation of actin filaments in podosomes was incomplete and residual actin filaments were observed in the cytoplasm (Fig. 4j), whereas Fak and Src were almost completely localized to podosomes ( Fig. 4l,n). These findings suggest that Cas participates in the transforming processes of Src through assembling actin filaments and that Cas-mediated actin aggregation in podosomes may be required for a-Src to induce a fully transformed phenotype.

Table 1 Colony formation in soft agar
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In this report, we have demonstrated that: (i) Cas is essential in organization of myofibrils and formation of Z-disks, which seems to be required for normal heart development and maintenance of blood vessel integrity during embryogenesis; (ii) Cas contributes to cytoskeletal organization through assembly of actin filaments; and (iii) Cas is required for Src-induced transformation, possibly through forming actin aggregates in podosomes. These results have provided an example of an adaptor molecule that maintains cytoskeletal organization and is pivotal in embryonic development and in oncogene-mediated transformation.

Methods

Construction of the targeting vector and generation of Cas-deficient mice.

Mouse Crkas was isolated from a mouse genomic library prepared from 129/SvJ mice DNA (Stratagene) by hybridization with the XhoI- BstXI fragment of rat Crkas cDNA (nt 134–440; ref. 1). A HindIII-HindIII genomic fragment was subcloned into pBluescript (Stratagene) and a neomycin resistance gene (Neo) derived from pMC1neopolyA (Stratagene) was inserted into an Xho I site existing in an exon encoding part of the SH3 region. The diphtheria toxin-A gene (DTA) from pMC1DTApolyA (Stratagene) was attached to the 5´ end of the targeting vector for negative selection. CCE ES cells (a gift of E. Robertson) were electroporated with NotI-linearized targeting vector and selection (250 μg/ml G418) was started 48 h after electroporation. Correctly targeted ES cells identified by Southern blot with 5´ and 3´ probes were injected into C57BL/6 blastocysts. Chimaeric males were mated with C57BL/6 females and heterozygous offspring were intercrossed to produce homozygous mutants. For genotyping, genomic DNA extracted from whole embryo was digested with BamHI and hybridized with 5´ and 3´ probes.

Histological analysis and immunohistochemistry.

Embryos were fixed in 4% paraformaldehyde, embedded in paraffin, serially sectioned in sagittal and parasagittal planes (4 μm), and stained with haematoxylin and eosin (H&E). Sections (500–1000 pieces/embryo) were carefully examined to detect histological differences between wild-type and Cas-deficient embryos. For immunohistochemistry, sagittal sections of wild-type embryos were dewaxed in xylene, rehydrated through a graded series of alcohols, treated with 0.3% H2O2 and incubated with anti-Cas antibody1. Positive signals were developed with diaminobenzidine substrate using the avidin-biotin-peroxidase system.

Electron microscopic analysis.

Hearts were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide in phosphate buffer, dehydrated through a graded alcohol series and embedded in Epon 812. Semi-thin sections were stained with toluidine-blue and the posterior wall of the left ventricle of each specimen was subjected to ultrathin sectioning and examination with a JEX2000 electron microscope after double-staining with uranyl acetate and lead citrate.

Culture of primary fibroblasts.

Primary fibroblasts prepared from embryos were maintained in Dulbecco's modified Eagle's medium (DMEM, supplemented with 10% fetal calf serum, 100 units/ml penicillin and 50 μg/ml streptomycin).

Immunofluorescence.

Hearts were frozen in OCT compound in liquid nitrogen. Primary fibroblasts were fixed with 3.7% formaldehyde in PBS and permeabilized with 0.2% Triton-X in PBS. Specimens were stained with antibodies and observed under a confocal microscopic system, MRC1024 (BioRad). The dilutions of primary antibodies were: 1:200 for anti-Cas (ref. 1), 1:200 for anti-actinin (Sigma), 1:200 for anti-vinculin (hVIN-1, Sigma), 2.5 μg/ml for anti-Fak (Takara) and 1:50 for anti-Src (MAb327; ref. 28). To visualize actin filaments, FITC-phalloidin and TRITC-phalloidin (Sigma) were used.

Transient expression of Cas in Cas-deficient cells.

Rat Crkas cDNA (ref. 1) was subcloned into an expression vector, pSSRα, and resultant plasmid was transfected into Cas-deficient fibroblasts using Superfect (Qiagen). After 48 h, cells were analysed using immunofluorescence.

Establishment of primary fibroblasts expressing a-Src and Cas.

Activated Src with Myc-tag (a-Src) was created by replacing aa 527–536 of rat Src, which include the negative-regulatory tyrosine residue, by c-Myc epitope tag (TSVDEQKLISEEDLN). For establishing a-Src-expressing stable transformants, a-src cDNA was subcloned into pSSRα and the resultant plasmid was transfected into wild-type and Cas-deficient cells with a plasmid containing a puromycin-resistance gene (10:1). For establishing a-Src-expressing, Cas-re-expressing cells, rat Crkas cDNA (ref. 1) was subcloned into an expression vector, bsr/pSSRα, which contains a blastocydine-resistance gene, and the resultant plasmid was transfected into a-Src-expressing, Cas-deficient cells. Transfection was performed using Lipofectamine (Gibco) according to the manufacturer's instructions and selection (1 μg/ml puromycin or 5 μg/ml blastocydine) was started 48 h after transfection. In each transfection experiment, approximately 30 independent antibiotic-resistant colonies were randomly picked up and subjected to biochemical analyses.

Preparation of cell lysates, immunoprecipitation and western blotting.

Proteins were extracted by homogenizing mouse embryos or lysing cultured cells in the RIPA lysis buffer1. For immunoprecipitation, protein aliquots were incubated with anti-Cas (1:500; ref. 1) coupled with protein-A. Samples were separated by SDS-PAGE, blotted with primary antibodies and positive signals visualized using the ProtoBlot Western AP system (Promega). The dilutions of primary antibodies were: 1:2500 for anti-Cas1, 5 μg IgG/ml anti-Myc (9E10; ref. 29) and 5 μg IgG/ml for anti-phosphotyrosine (4G10; ref. 30).

Colony formation in soft agar.

Cells were suspended in DMEM+20% FCS and 0.3% agar and were layered on a bottom layer containing 0.6% agar. Numbers of colonies were counted 2 weeks after plating.