QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online August 3, 2005
doi: 10.1242/10.1242/jcs.02476

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Dorner, A. A. Articles by Vestweber, D. Search for Related Content PubMed PubMed Citation Articles by Dorner, A. A. Articles by Vestweber, D. Social Bookmarking                         What's this?

Coxsackievirus-adenovirus receptor (CAR) is essential for early embryonic cardiac development

Armin A. Dorner^1,*, Frank Wegmann^2,*, Stefan Butz², Karen Wolburg-Buchholz³, Hartwig Wolburg³, Andreas Mack³, Ines Nasdala², Benjamin August², Jürgen Westermann⁴, Fritz G. Rathjen¹ and Dietmar Vestweber^2,5,

¹ Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Robert-Rössie-Straße10, 13125 Berlin, Germany
² Institute of Cell Biology, ZMBE, University of Münster, 48149 Bad Nauheim, Germany
³ Institute of Pathology, University of Tübingen, Wilhelmstr. 7, 72074 Tübingen, Germany
⁴ Institute of Anatomy, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany
⁵ Max-Planck-Institute of Molecular Biomedicine, Von-Esmarch-Straße 56, 48149 Münster, Germany

View larger version (38K): [in a new window]   Fig. 1. Targeted disruption of the CAR gene in mice. (A) Targeting strategy. A map of the relevant genomic region containing the first ATG-containing exon of CAR (top), the targeting vector (middle) and the mutated locus after recombination (bottom) are shown. Dor25, GS3 and Neo2L represent oligonucleotides used for PCR screening. The positions of probes used for Southern screening are depicted as bars #1 and #2. E=EcoRV, X=XhoI. (B) Detection of wild-type and targeted alleles by Southern blot analysis. Genomic DNA of mice of the F1-generation was digested with EcoRV and XhoI and hybridized with the combination of probes #1 and #2. DNA of wild type (+/+) mice gave an 8 kb signal (EcoRV fragment), +/– heterozygotes gave an additional signal for the 6.4 kb EcoRV/XhoI fragment. (C) PCR analysis of genomic DNA with the oligonucleotides Dor25, GS3 and Neo2L described in A. A 1kb product is generated from the wild-type allele and a 1.5 kb product from the targeted allele. (D) Western blot of SDS-PAGE extracts of mouse embryos (E11.5) of the three different genotypes with antibodies against mouse CAR. Note that heterozygotes expressed less CAR protein than homozygous wild-type embryos. (E) Activity and specificity of affinity purified rabbit anti-mouse CAR antibodies illustrated by western blot analysis of mock transfected CHO cells and CAR-transfected CHO cells (as indicated). Molecular mass markers are indicated on the left. (F) Specificity control for affinity purified anti-CAR antibodies (VE15) used for indirect fluorescence staining of the heart region of E11.5 CAR+/+ and CAR-/- embryos (as indicated).

View larger version (134K): [in a new window]   Fig. 2. Analysis of the vascularization of CAR-deficent embryos. Wild type (+/+) and CAR-deficient (–/–) embryos of stage E10.5 (top) or E11.5 (middle) were stained as whole mounts with anti-PECAM-1 antibodies. The head region is shown for E10.5, limb buds are shown for E11.5. No obvious differences in overall vascularization were observed. Bottom panel: paraffin sections of E11.5 embryos. Sections were taken from the posterior limb region, and stained with hematoxylin and eosin (H+E). Note: aortae and cardinal veins were enlarged in CAR-/- embryos. Key: a, left and right aorta; c, left and right cardinal vein. Bar, 200 µm. In each case, CAR+/+ and CAR-/- littermates were compared.

View larger version (68K): [in a new window]   Fig. 3. Analysis of placental expression of CAR and of overall placenta organization of CAR-deficient embryos. (A) Paraffin sections of wild type (+/+) and CAR-deficient (–/–) placentae (E11.5) were stained with antibodies against the endothelial marker endomucin and counterstained with hematoxylin. Since maternal vessels are devoid of endothelial cells the endothelial staining shows selectively only blood vessels of the fetus. The different zones of the placenta are marked: gl, giant layer; s, spongiotrophoblast layer; l, labyrinthine layer; cp, chorionic plate. Note: the extension of the labyrinthine layer was not altered in CAR-/- embryos. Bar, 100 µm. (B) Cryostate section of a placenta of E11.5 wild-type embryo double stained with anti-CAR polyclonal antibodies (red) and an anti-PECAM-1 mAb (green) visualized by indirect immunofluorescence. Nuclei were visualized with Systox (blue). CAR was found on trophoblast cells of the labyrinth zone, but not on endothelial cells. Bar, 20 µm.

View larger version (156K): [in a new window]   Fig. 4. CAR is not expressed on endothelial cells or vascular smooth muscle cells of E11.5 embryos. Cryostate sections of E11.5 embryos were double stained with antibodies against CAR (green), and either ESAM or smooth muscle actin (SMA) as indicated (red). The expression patterns of the endothelial marker protein ESAM and of SMA were non-overlapping with the distribution pattern for CAR. Key: a, aorta; lu, developing lung buds; p, pericard; m, myocard; c, cardinal vein; al, atrial lumen; vl, ventricular lumen; oe, oesophagus; tr, trachea. Bar, 100 µm.

View larger version (96K): [in a new window]   Fig. 5. Abnormalities of the heart of CAR-deficent embryos. (A) Whole mounts of wild type (+/+) and CAR-deficient (–/–) E11.5 embryos of the same litter are shown. The asterisk marks the enlarged pericard in the CAR-/- embryo. Hemorrhages resulting from preparing the embryos were more often observed in CAR-/- embryos than in wild-type embryos. (B) Paraffin sections of E11.5 embryos of the same litter were counterstained with hematoxylin. The micrographs show the heart region. CAR-/- embryos showed enlarged endocardial cushions and a single persistent atrioventricular canal instead of two canals in the wild-type embryo. Key: a, atrium; v, ventricle; e, endocardial cushion; *, atrioventricular canal. Bar, 100 µm.

View larger version (112K): [in a new window]   Fig. 6. No signs for altered proliferation or apoptosis activity in cardiomyocytes of CAR-deficient embryos. Upper row: 2 hours before preparing of E11.5 embryos, the pregnant mother was injected with BrdU. Cryostate sections of CAR+/+ and CAR-/- littermate embryos were stained for incorporated BrdU (red) and counterstained for sarcomeric -actinin (blue) to mark cardiomyocytes. Micrographs show the area of the heart ventricles. For better visualization of the red staining, the lightness of the blue staining was enhanced. Bar, 100 µm. Bottom row: detection of apoptotic cells by TUNEL staining (green) of cryostate sections of E11.5 littermate embryos. Cardiomyocytes were stained for sarcomeric -actinin (red). No significant signs of apoptosis were detected independently of the genotype. Bar, 200 µm.

View larger version (109K): [in a new window]   Fig. 7. Alterations of cardiomyocytes in CAR-deficient embryos. Ultrathin sections of the heart of E11.5 CAR+/+ (left) and CAR-/- (right) littermate embryos (A-D) were analyzed by transmission electron microscopy. The micrographs show alterations within cardiomyocytes, such as enlarged mitochondria (M), mitochondria with increased electron density (*). Most importantly large areas of the cytosol of CAR-/- cardiomyocytes were devoid of myofilament bundles and the thickness of these bundles was much reduced (arrows). Bar in A and B represents 0.5 µm, and in C and D 1 µm. (E,F) Semi-thin sections of E11.5 embryos of both genotypes were prepared and the heart region was stained for the accumulation of glycogen granula by periodic acid Schiff staining. Note that CAR-/- embryos showed a strong increase in glycogen storage. Bar, 25 µm.

View larger version (92K): [in a new window]   Fig. 8. Comparison of in vitro cultured wild type and CAR-/- embryonal cardiomyocytes. Cardiomyocytes of E11.5 embryos isolated and cultured for 3 days were stained by indirect immunofluorescence with antibodies against sarcomeric -actinin. Three examples of typical wild-type cells (+/+) and CAR-deficient cells (–/–) are depicted. Note: the myofibrils were disorganized and shortened in CAR-/- cells.

View larger version (79K): [in a new window]   Fig. 9. Orientation and localization of myofibrils in the vicinity of cell contacts and focal contacts in embryonal CAR+/+ and CAR-/- cardiomyocytes. (A) Indirect immunofluorescence of CAR+/+ cells (+/+) for CAR and of CAR+/+ cells and CAR-/- cells (–/–) for ZO-1. (B-D) Double staining of CAR+/+ and CAR-/- cells for: (B) N-cadherin (green) and ß-catenin (red); (C) ß1-integrin (green) and -actinin (red); (D) N-cadherin (green) and -actinin (red). In B and C the single stainings and the merge are shown (as indicated) whereas in D only the merged picture is depicted. Bars in A-C represent 50 µm, bar in D is 10 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter