Vascular cell adhesion molecule (VCAM-1) was originally identified as a cytokine inducible surface protein that mediated adhesion of a number of leukocytes ...
Development 121, 489-503 (1995) Printed in Great Britain © The Company of Biologists Limited 1995
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Defective development of the embryonic and extraembryonic circulatory systems in vascular cell adhesion molecule (VCAM-1) deficient mice Lia Kwee1,*, H. Scott Baldwin2,3,*, Hong Min Shen2, Colin L. Stewart4, Catherine Buck2, Clayton A. Buck2 and Mark A. Labow1,† 1Department of Biotechnology, Roche Research Center, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley NJ 07110-1199, USA 2Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19103-4268, USA 3Division of Pediatric Cardiology, Children’s Hospital of Philadelphia, Philadelphia, PA, 19026, USA 4Roche Institute of Molecular Biology, 340 Kingsland Street, Nutley NJ 07110-1199, USA
*These authors contributed equally to the work †Author for correspondence
SUMMARY VCAM-1 is a cytokine-inducible cell surface protein capable of mediating adhesion to leukocytes expressing α4 integrins. Mice deficient in VCAM-1 expression were produced by targeted homologous recombination in ES cells. VCAM-1-deficient embryos were not viable and exhibited either of two distinct phenotypes. Approximately half of the embryos died before embryonic day 11.5 and exhibited a severe defect in placental development in which the allantois failed to fuse with the chorion. The remaining VCAM-1-deficient embryos survived to embryonic day 11.5-12.5 and displayed several abnormalities in their developing hearts including a reduction of the compact layer of the ventricular myocardium and intraventricular
septum. The hearts also contained significant amounts of blood in the pericardial space and lacked an epicardium. α4 and VCAM-1 were found to be expressed in wild-type embryos in a reciprocal fashion in the chorion and allantois and in the epicardium and the underlying myocardium, although VCAM-1 was expressed in the intraventricular septum in the absence of adjacent α4-expressing cells. These data suggest important roles for VCAM-1 and α4 in the development of the placenta and the heart.
INTRODUCTION
encoded by alternatively spliced mRNAs. For example, mRNA encoding a 6-domain form of human VCAM-1 lacking the fourth Ig-domain (Osborn et al., 1992; Polte et al., 1991) and alternatively spliced rabbit VCAM-1 mRNAs encoding additional Ig-domains have been identified (Cybulsky et al., 1991). These isoforms interact with α4 integrins, although their functional significance is not known at this time. A unique isoform, VCAM-1GPI, has been identified in mice (Moy et al., 1993; Terry et al., 1993). VCAM-1GPI contains only the first three Ig domains and is anchored to the cell membrane via glycosylphospatidlyinositol. VCAM-1GPI is encoded by an alternatively spliced mRNA that is preferentially induced by inflammatory cytokines and LPS. Interestingly, the VCAM-1 gene of both humans and mice is organized such that each Ig domain is encoded by a single exon with each exon terminating in the same reading frame, facilitating the production of multiple protein isoforms from alternatively spliced mRNA (Cybulsky et al., 1991, 1993; Terry et al., 1993). VCAM-1 function has been associated primarily with whitecell endothelial cell interactions in response to inflammatory cytokines and with early T-cell and B-cell maturation. It is intimately involved in inflammatory processes such as those associated with arthritis, allograft rejection, and atherosclerosis
Vascular cell adhesion molecule (VCAM-1) was originally identified as a cytokine inducible surface protein that mediated adhesion of a number of leukocytes including lymphocytes, monocytes, mast cells, eosinophils and tumor cells to umbilical vein endothelial cells (Osborn et al., 1989; Rice and Bevilacqua 1989; Bochner et al., 1991; Carlos et al., 1991). VCAM1 is a type 1 transmembrane protein belonging to the immunoglobulin superfamily. Structurally, the extracellular amino-terminal portion of the molecule consists of seven immunoglobulin- (Ig) like domains followed by a typical hydrophobic transmembrane domain and terminating in a short cytoplasmic domain. The Ig-like domains appear to have arisen by internal duplication, as domains I,II and III are highly homologous to domains IV, V, and VI (Polte et al., 1991). Both Ig-domains I and IV are capable of independently interacting with α4 integrins, the only known counter receptors for VCAM-1 (Elices et al., 1990; Osborn et al., 1992; Vonderheide et al., 1994; Ruegg et al., 1992). Recent evidence suggests that binding to domain IV may require activation of the leukocyte integrins (Needham et al., 1994). Multiple VCAM-1 isoforms have been identified that are
Key words: vascular cell adhesion molecule, mouse, placental development, cardiogenesis
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(Cybulsky and Gimbrone, 1990; van Dinther-Janssen et al., 1991; Brockmeyer et al., 1993; Ferran et al., 1993). AntiVCAM-1 monoclonal antibodies have been found to reduce the severity of experimentally induced autoimmune encephalitis and to prevent the rejection of cardiac allografts (Pelletier et al., 1992; Orosz et al., 1993). VCAM-1 may also enhance Tcell reactivity through its ability to act as a co-stimulatory molecule, facilitating the activation of antigen-specific T-cells (Damle and Aruffo, 1991; van Seventer et al., 1991). VCAM1 has also been implicated in B-cell maturation and hematopoiesis. Murine VCAM-1 is expressed in follicular dendritic cells of secondary lymphoid organs and in murine bone marrow stromal cells (Freedman et al., 1990; Miyake et al., 1991). Antibodies to murine VCAM-1 will block the development of B-cells in long term bone marrow cultures, presumably by preventing direct B-cell/stromal cell binding. Recent observations suggest that VCAM-1 function may also be important in embryonic development. VCAM-1 is expressed on secondary myocytes and myotubes during skeletal muscle development and the α4β1 integrin is expressed on forming myotubes (Rosen et al., 1992; Stepp et al., 1994). Antibodies against either of these molecules block myotube formation in culture. Immunohistochemical studies of VCAM1 and α4 integrins in embryonic day (ED) 13 to 15 mouse embryos revealed a wider distribution of both molecules than would be expected if their function was limited to hematopoiesis and skeletal muscle development (Sheppard et al., 1994). While α4 integrins were more widely distributed than VCAM1, likely reflecting their ability to recognize either VCAM-1 or fibronectin (Elices et al., 1990), VCAM-1 was detected in the myocardium of the heart, particularly in regions of the atrioventricular septum and the outflow tract. α4, however, was detected in the epicardium enclosing the heart and the endocardial cushions. Both molecules were expressed in the yolk sac at sites of hematopoiesis, with α4 expression also extending to the smooth muscle cells surrounding the vitellin vessels. α4, but not VCAM-1 was detected in the chorionic plate of the developing placenta in the 9 day mouse embryo. In order to evaluate the roles of VCAM-1 in mouse development and inflammatory disease, a null mutation has been introduced into the VCAM-1 gene in embryonic stem (ES) cells, and the targeted cells used to produce VCAM-1-deficient mice. Mice homozygous for the VCAM-1 mutation were not viable, and died between 10.5 and 12.5 days post coitum (dpc). VCAM-1-deficient embryos displayed two distinct phenotypes. One group of embryos failed to develop a functional extraembryonic circulation due to the failure of the allantois to fuse with the chorion. The other group displayed several defects in the developing heart, suggesting that they may have subsequently died from abnormal cardiac function. In addition, VCAM-1 and the α4 subunit of the known VCAM-1 counter receptors were found to be expressed in a reciprocal fashion in tissues affected by the VCAM-1 mutation with the exception of the intraventricular septum, where high levels of VCAM-1 expression were found in the absence α4 expression on adjacent cells. These data indicate that VCAM-1 and its counter receptor(s) mediate cell-cell interactions essential for normal mouse development. These observations have obvious implications concerning the potential dangers of the use of VCAM-1 and α4 antagonists during pregnancy.
MATERIALS AND METHODS Construction of the VCAM-1 targeting vector The targeting vector was produced as follows. All genomic subclones of the VCAM-1 gene were described previously (Terry et al., 1993). A 1.7 kb Spe-BglII fragment containing exon one through half of exon two (encoding domain I) was isolated from the genomic subclone pBST2Bam and inserted into the BamHI site of pPGKneotk which was a generous gift of Dr Thomas Gridley. The resulting plasmid, pPGKneotk/1.7 was then digested with ClaI and treated with the Klenow fragment of DNA polymerase I (New England Biolabs, Inc) in the presence of all four deoxyribonucleotides. Finally, an 8.5 kb SalI-StuI fragment from the VCAM-1 genomic clone, pBSVCAM19, was inserted into the SalI-ClaI digested pPGKneotk/1.7. DNA for ES cell transformation was purified using a Quiex column (Quiagen Inc.) and subsequently linearized with SalI. ES cell transformation and production of chimeric mice The ES cell line, W9.5, was cultured in the presence of irradiated primary embryonic fibroblasts. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (JRH Biosciences, inc.) containing 2 mM glutamine, 15% fetal calf serum (Gibco-BRL Inc.) 1× nonessential amino acids (Gibco-BRL Inc), 0.1 mM β-mercaptoethanol (Sigma, Inc) and 1000 U/ml LIF (Gibco-BRL, Inc) at 37°C and in air containing 10% CO2. ES cell culture and transformation were carried out essentially as previously described (Stewart et al., 1992; Abbondanzo et al., 1993). Approximately 4.5×107 ES cells were chilled on ice for 5 minutes and then electroporated at room temperature with 25 µg of the linearized targeting vector in 700 µl of PBS. After electroporation, the ES cells were passaged into plates containing irradiated feeder cells. The next day (day 1) G418 was added to the medium to a final concentration of 400 µg/ml. On day 2, the cells were refed with medium containing G418 and 0.2 µM FIAU, in order to select for neo+tk− cell clones according to the positive-negative selection strategy (Mansour et al., 1988). Selection continued for 8 more days with daily replacement of medium. On day 10, surviving clones were isolated and plated on a layer of fresh feeder cells in individual wells of a 48-well dish. DNA form the individual W9.5 clones was isolated essentially as described by Laird et al. (1991). Half of the isolated DNA was digested with BglII (New England Biolabs, Inc.) fractionated by agarose gel electrophoresis and transferred to nylon filters (Biotrans, ICN, Inc.). The filters were then analyzed by hybridization simultaneously to two 0.65 kb DNA fragments shown in Fig. 1. The two probes correspond to regions just on both sides of the 5′ VCAM-1 fragment contained in the targeting vector. Radiolabeled probes were produced using the Prime It II-kit (Stratagene, Inc.). All hybridization was carried out in Church buffer (Church and Gilbert, 1984) at 65°C and filters were washed by standard procedures. ES cell clones carrying a disrupted VCAM-1 gene were used to produce chimeric mice by injection into the blastocoel cavity of 3.5day C57BL/6J embryos as previously described (Bradley, 1987). The injected blastocysts were implanted into 2.5-day psuedopregnant female mice. Male chimeric mice were then bred with C57BL/6J females to determine germline chimerism and to isolate heterozygous animals for breeding. Timed matings were carried out by assigning the morning of identification of vaginal plugs as day 0.5. Preparation of embryos Yolk sacs from embryos were used to make genomic DNA for genotyping. All genotyping of embryos was carried out by Southern blot analysis as described above. Embryos from timed matings were carefully dissected and placed in freshly made 4% paraformaldehyde in PBS and incubated at 4°C overnight. The embryos were then placed in fresh paraformaldehyde for 30 minutes at 4°C followed by sequential 15 minute incubations in 1:1 70% ethanol:PBS, 70% ethanol (two times) and then stored in 70% ethanol. For paraffin wax sections the
VCAM-1-deficient mice 70% ethanol was replaced sequentially, for 30 minutes each with, 85% ethanol, 95% ethanol, twice in 100% ethanol and twice in xylene. Finally the embryos were incubated in a 1:1 xylene:paraffin wax at 60°C overnight then three times in fresh paraffin wax before embedding in pure, fresh paraffin wax. For tissue preparation for expression analysis, timed, pregnant CD1 mice were purchased from Harlan Sprague Dawley, Inc. Embryos of various gestational ages were removed by cesarean section, dissected free of the desidual mass and staged according to the method of Kaufman (Kaufman, 1992). Embryos used for whole-mount immuohistochemistry were washed three times in Tris-buffered saline, pH 7.4 (TBS), after brief fixing in Dent fixative and immediately processed. Embryos used for cross sectional immunohistochemistry were processed through a sucrose gradient in phosphate-buffered saline embedded in OCT and frozen in liquid nitrogen-cooled isopentane and stored at −70°C until used. Whole-mount immunohistochemistry Our procedure for whole embryo immunolabelling closely followed that of Davis (1993). Briefly, primary and secondary antibodies were diluted in 4% bovine serum albumin in TBS. Embryos were exposed to the primary antibody overnight at 4°C. The following day they were passed through 5 hourly changes of TBS and subsequently counter stained overnight with horseradish peroxidase-conjugated goat antirat secondary antibody diluted 1:200. Following 5 hourly changes of TBS, antibody binding was detected using diaminobenzidine hydrochloride substrate for up to 4 hours. The reaction was terminated in methanol and the stained embryo cleared in benzyl alcohol:benzyl benzoate (2:1). Specimens were mounted in deep well glass slides and photographed on a Nikon SMZ-U microscope. Cross sectional immunofluorescent staining 7 µm cryostat sections of embedded embryos were fixed in methanol at −4°C and stored at −20°C. They were rehydrated in PBS for 30 minutes, soaked in a solution containing 4% BSA and 4% goat serum in PBS for 30 minutes and exposed to the appropriate primary monoclonal antibody for 1 hour in a humidified chamber at room temperature. The slides were washed three times in PBS and then counterstained with a mixture of FITC-conjugated goat anti-rat secondary antibodies in a humidified chamber at room temperature in the dark for 1 hour. The specimens were washed three times in PBS, mounted in antiquench mixture containing DABCO and photographed using a Leica fluorescence microscope. Both antibodies used were goat antirat IgG and included: anti-α4, 9C10 (Pharmingen N0. 4874) at 50 mg/ml and anti-VCAM-1, 429 (Pharmingen No. 5741) at 30 mg/ml.
RESULTS Targeted disruption of the murine VCAM-1 gene The structure of the murine VCAM-1 gene, as previously described, is illustrated in Fig. 1. The genomic organization, as described above, suggested that introduction of a simple frame shift mutation could still allow the production of functional VCAM-1 protein if the mutated exon was removed by alternative mRNA splicing. Therefore, a targeting vector was designed to introduce a frame shift and a deletion mutation into the VCAM-1 locus in order to prevent the production of any functional protein by fortuitous splicing events. A PGKneo resistance cassette was inserted within the coding region of the first Ig domain (exon 2) and flanked by sequences downstream of the exon encoding domain IV (exon 5). Homologous recombination between this vector and the VCAM-1 locus should introduce a frameshift mutation in the middle of domain I and delete the remainder of domain I, the exon 2 splice donor and
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genomic sequences encoding domains II, III and IV. Thus, the coding region of all Ig domains essential for α4 integrin binding would be disrupted and/or deleted from the targeted allele. This vector was used to transform W9.5 ES cells and neor/FIAUr colonies were isolated. Southern blot analysis of genomic DNA digested with BgIII and hybridized to genomic DNA probes identified multiple clones exhibiting a unique 5.2 kb BgIII band expected from homologous recombination with the construct as shown in Fig. 1B. The frequency of homologous recombination was approximately 30%. Additional Southern blot experiments were used to confirm the structure of the disrupted VCAM-1 locus (data not shown and see below). ES cell clones were karyotyped and those showing a normal 40XY karyotype were used to produce chimeric mice by blastocyst injection. Two of the three injected ES cell clones gave rise to highly chimeric males that transmitted the mutant VCAM-1 allele through the germline and were subsequently used for analysis. The absence of a functional VCAM-I gene results in embryonic death Heterozygous mice were intercrossed and their progeny genotyped. An example of the genotypic analysis of embryos is shown shown in Fig. 1C. DNAs shown are from 13.5 dpc homozygous wild-type (+/+), heterozgous (+/−) or homozygous mutant (−/−) embryos. In this experiment, a duplicate blot was also probed with a portion of the murine VCAM-1 cDNA consisting of the coding region for Ig-domains II-IV (exons IIIV) in order to confirm the structure of the predicted deletion mutation. As shown, hybridization to the cDNA probe was only detected from DNAs from the +/+ and +/− embryos demonstrating that embryos homozygous for the targeted mutation did not contain any copies of the deleted VCAM-1 sequences. Only one live animal, homozygous for the VCAM-1 mutation, was identified after screening almost 900 offspring from heterozygous intercrosses (Table 1). This single animal died at approximately 6 weeks of age and the body was Table 1. Genotypes of progeny from VCAM-1 heterozygote intercrosses Age (nos.=dpc)
+/+
+/−
−/−
Live births
299 (35%)
563 (65%)
1* (0.1%)
10.5
15 (25%)
32 (53%)
13 (22%) (6/13 small)
11.5
22 (26%)
39 (47%)
16 live† (19%) 7 dead‡ (8%)
12.5
21 (25%)
39 (47%)
3 live (2%) 21 dead‡ (25%)
13.5
18 (27%)
39 (65%)
11 dead‡ (8%)
*The one homozygous −/− adult died at 6 weeks of age and the remains were not available for analysis. Thus the genotype of this animal could not be verified. †Embryos were judged as live if they were normal in size and color and if the heart appeared to be beating. ‡Dead embryos were those that were smaller and whiter than live litter mates and did not contain a beating heart but were generally in good condition such as that in Fig. 6C. Some embryos were already undergoing resorption and were highly necrotic and contained no visible internal organs. All −/− embryos at 13.5 dpc were already undergoing resorption.
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Fig. 1. Disruption of the murine VCAM-1 locus. (A) Targeted disruption of the VCAM-1 gene. The top line shows the organization of the murine VCAM-1 gene. Noncoding regions of exons are indicated by open boxes and coding regions by black boxes. The exon number is indicated on the top and the location of exons encoding the signal peptide (S), Ig domains (I through VII), the transmembrane domain (Tm) and cytoplasmic domain (Cyt) are indicated below. The middle line represents the knockout construct. The PGKneo cassette is indicated by a stippled box and the MC1TK cassette used for negative selection is indicated by the checkered box. Plasmid vector sequences are indicated by the dashed line. The bottom line represents the predicted structure of the targeted VCAM-1 allele. Restriction sites indicated are those for BglII (Bg) and SpeI (Sp). Two probes were used simultaneously to type the mice, and indicated by small open boxes. The size VCAM-1 Locus of the BglII fragments detected by the 5 12 3 4 ∆5 6 7 8 9 Exon probes are indicated for the endogenous probes Bg Bg Bg Bg Sp allele and the targeted allele. (B) Identification of ES cells carrying a Domain S I II III IV V VI VII Tm/Cyt targeted VCAM-1 gene. ES cell DNA 2.6 kb 0.8 kb digested with BglII was analyzed by hybridization with the probes described above. The Southern blot also contains the original genomic subclone, pBAM.2 Bg PGKneo (pBAM.2; Terry et al., 1993) digested with BglII and the knockout construct vector MC1TK digested with BglII (KO) as positive and V VI VII Tm/Cyt SI negative controls, respectively. The Knockout Construct markers on the left represent 35S-DNA markers from Amersham Inc. The 2.6 kb and 0.8 kb bands derived from the WT VCAM-1 gene and the 5.2 kb band 5.2 kb specific for the disrupted VCAM-1 gene are indicated on the Bg PGKneo Bg Sp right. (C). Genotypic analysis of 13.5 dpc embryos derived from heterozygous intercrosses. DNAs from two independent embryos of each genotype are shown. The Domain S I V VI VII Tm/Cyt DNAs were digested with BglII and hybridized as described Structure of Disrupted Locus for B or with a probe derived from the murine VCAM-1 cDNA consisting of the coding region for domains II,III, and IV. This probe identifies a 9 kb DNA fragment from mouse genomic DNA that is absent from the DNA of mice homozygous for the targeted mutation.
A
unavailable for further analysis. Breeding cages were closely monitored to identify any pups that might have died perinatally, although no additional homozygous pups were identified. Thus, in our experiments, VCAM-1-deficient embryos were not viable. Embryos from timed heterozygous intercross matings were subsequently isolated on sequential dpc in order to determine the time of death of embryos homozygous for the VCAM-1 mutation (−/− embryos). As shown in Table 1, −/− embryos were found at approximately the expected Mendelian frequency (22%) at 10.5 dpc. Although all the −/− embryos appeared viable at this time, half were noticeably smaller than the wild-type (VCAM- +/+ embryos; WT) and heterozygous littermates. At 11.5 dpc, 25% of the embryos were identified
as −/−. At this time, however, a third of the −/− embryos had already died, were highly necrotic and were undergoing resorption. The size of the dead embryos at 11.5 dpc suggested that they had failed to develop past 10.5 dpc (data not shown and see below), although their poor condition precluded a more detailed analysis. Most of the VCAM-1 −/− embryos (13 out of 19 11.5 dpc embryos analyzed) appeared viable and displayed no obvious gross morphological differences compared to their heterozygous and WT litter mates. No −/− embryos survived beyond 12.5 dpc. The majority of VCAM-1 −/− embryos identified at 12.5 dpc appeared to be undergoing resorption. Three −/− embryos examined at this time appeared viable, although two of these were significantly paler than het-
VCAM-1-deficient mice Fig. 2. Expression of VCAM-1 and α4 in early postimplantation embryos. (A-C) Whole-mount immunohistochemical analysis of VCAM1 expression. Embryos isolated at either ED 7.5, 8.75 or 9.5 were stained with an antibody to murine VCAM-1 as described in the text. The location of the heart (h), allantois (a) and somites (s) are indicated with arrows. Control experiments carried out without the primary antibody to murine VCAM-1 are included (−ab). The locations of myocardial cells expressing VCAM-1 in A are indicated by small arrows, while the underlying abdominal intestinal pocket (aip) is indicated with a large arrow. D and E represent immunofluorescence analysis of a sagittal section of an ED 8.5 embryo stained with an antibody to murine VCAM-1 or α4, respectively. The locations of the allantois (a) and the chorion (c) are indicated with arrows. The arrowheads indicating the chorion have been placed immediately on top of the continuous chorionic membrane to mark precisely its location.
erozygous or WT litter mates. At 13.5 dpc, all homozygous null embryos were dead and at various stages of necrosis or resorption. These data demonstrate that most VCAM-1-deficient embryos died between 10.5 and 12.5 dpc. These embryos may also represent two distinct groups, one being affected by 10.5 dpc and dead by 11.5 dpc and a second, remaining viable until 11.5 or 12.5 dpc. Expression of VCAM-I and α4 integrins in early postimplantation embryos Interpretation of the possible origin of developmental abnormalities leading to the death of the homozygous null embryos requires the elucidation of the expression pattern of VCAM- 1 and α4 integrins prior to embryonic death. This was examined by immunohistochemical staining of whole-mount 7.5 to 9.5 dpc WT embryos using antibodies against murine VCAM-1 (Fig. 2). These experiments revealed that VCAM-1 was expressed at two major sites within the embryos, the distal end of the developing allantois and the heart. VCAM-1 was detected within the developing heart from the earliest time of its morphogenetic organization (Baldwin et al., 1991) through at least 13.5 dpc (Figs 2A, 3E and data not shown), suggesting a possible role of VCAM-1 in heart development. Examination of sequential sagittal sections of 8.5 dpc WT embryos,
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VCAM
α4
ED 11.5
ED 8.75
Myosin
Fig. 3. Expression of VCAM-1 and α4 in ED 8.75 and ED 11.5 hearts. Cross sections of embryos were analyzed by immunofluorescence after staining with antibody to the alpha chain of myosin (A,D), VCAM-1 (B,E), or α4 (C,F), as described in the text. Specific structures are indicated as follows; neural tube (nt), fore gut (fg), endocardium (e) myocardium (m), right ventricle outflow tract (rvot), endocardial cushion (edc), right ventricle (rv), left ventricle (lv), and intraventricular septum (ivs). The location of the epicardial expression α4 is indicated by arrows in F.
including the extraembryonic membranes, confirmed the localized expression of VCAM-1 to the distal region of the allantois (Fig. 2D). In addition, VCAM-1 was found in the mesenchyme of the forming yolk sac at this time (data not shown). Staining for α4 showed that the VCAM-1 counter receptor was not expressed at this time in the heart, but was readily detected in the chorionic epithelium juxtaposed to the VCAM-1-expressing regions of the allantois (Fig. 2E) as well as in complementary positions within the developing yolk sac
(data not shown). Thus, VCAM-1 and α4 were expressed specifically within the distal region of the advancing allantoic stalk and the chorion at least 48 hours prior to the time of their fusion and the establishment of the interface between embryonic and maternal circulation, suggesting that the counter-receptors may contribute to this process. The expression of VCAM-1 and α4 in the heart was examined in adjacent sections of 8.75 and 11.5 dpc embryos, as this was the second major site of VCAM-1 expression. Fig. 3 shows
VCAM-1-deficient mice neighboring sections stained with an antibody to alpha myosin to more clearly delineate the myocardium. In the 8.75 dpc embryo, VCAM-1 expression was observed throughout the myocardium of the forming heart tube. The outer myocardial layer showed diffuse expression of VCAM-1 in the absence of α4 expression. The inner endothelial cells making up the endocardium, well separated from the myocardium by a thick extracellular matrix or cardiac jelly, expressed neither VCAM- 1 nor α4 at this stage. By 11.5 dpc, heart morphogenesis has advanced to the point that the forming chambers of the mature structure are readily discernible (Fig. 3D-F). The ventricular myocardium is clearly divided into an outer, compact layer of dividing myocardial cells; the more delicate trabeculae, extending into the chamber of the septating ventricle and finally, the intraventricular septum that will eventually separate the right and left ventricles. At this time, α4 expression was observed in the single layer of epicardial cells encasing the myocardium (Fig. 3F) adjacent to the underlying compacted layer of the myocardium which, along with the intraventricular septum, showed a high level of VCAM-1 expression. The presence of α4 protein was also observed in the mesenchymal cells derived by epithelial-mesenchymal transformation of overlying endothelial cells that have migrated into the forming endocardial cushion (Markwald et al., 1975). However, no VCAM-1-expressing cells were found within the endocardial cushions. Embryos homozygous for the VCAM-1 mutation produce no detectable VCAM-1 protein Expression of VCAM-1 and α4 was also examined in the hearts of VCAM1 −/− embryos at 11.5 dpc. An example of these studies is shown in Fig. 4. The location of the myocardium is indicated by staining with antibody to alpha myosin (Fig. 4A). The presence of an intact endocardium is also indicated by staining for PECAM-1, an Ig-superfamiliy member that is expressed in all endothelial cells (Albelda et al., 1990; Muller et al., 1993; Baldwin et al., 1994). Staining of adjacent heart sections with antibody to VCAM-1 failed to detect any VCAM-1 protein. Expression of α4 was still detected in the endocardial cushions, although α4expressing epicardial cells were no longer seen (Fig. 4D). It is unclear if
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this cell layer is absent or simply no longer expressing α4, as the epicardium consists of only a single layer of cells that cannot be clearly resolved on frozen sections in the absence of a positive fluorescence signal. Thus, no detectable VCAM-1 protein was produced by embryos homozygous for the targeted mutation, indicating that the targeted allele contains a null
Fig. 4. No VCAM-1 protein can be detected in the heart of an embryo homozygous for the VCAM-1 mutation. The heart of a VCAM-1 −/− embryo was stained with antibody for the alpha chain of myosin, PECAM, VCAM-1 or Author for correspondenceα4 as indicated (A, B,C and D, respectively). Structures indicated are the endocardial cushions (c), right ventricle (rv), left ventricle (lv), intraventricular septum (s), and endocardium (e).
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Fig. 5. Failure of the allantois to fuse with the chorion in VCAM-1-deficient embryos. Examples of WT (+/+) and VCAM-1-deficient (−/−) embryos isolated at 9.5 dpc are shown. The genotypes of the embryos are shown in the upper right hand corner of each photograph. The allantois in each case is indicated by an arrow except in D and E. The embryos shown in A and C were photographed before removal of the extraembryonic membranes. A and B are pictures of the same embryo. The allantois is clearly fused to the placenta in the WT embryo in A and C and small vessels are clearly observed within the allantois. C,D,and E are the same embryo at different magnifications. The homozygous null embryo shown in F contained a small allantois that was not fused to the chorion. The embryo in G contains an allantois that is fused to the placenta on its side rather than the distal end such as that B. All embryos were approximately the same size and were taken at different magnifications in order to fill the frame for photographic purposes.
mutation. This conclusion is consistent with Southern blot and PCR experiments which confirmed the deletion of the coding region for Ig domains I, II,III, and IV (Fig. 1 and data not shown). Chorion-allantois fusion in VCAM-1-deficient embryos The reciprocal pattern of expression of VCAM-1 and α4
observed in the allantois and the chorion suggested that these molecules might be involved in placentation. To investigate the potential role of VCAM-1 in this process, staged embryos, derived from heterozygous intercrosses, were examined in detail. The earliest phenotype observed in VCAM-1 −/− embryos was, indeed, the absence of allantois-chorion interconnection (Fig. 5). The allantois is a mesodermally derived structure that grows out from the hind gut of the embryo,
VCAM-1-deficient mice eventually forming a connection with the placenta by fusion with the chorion, giving rise to the chorioallantois (for review see Steven and Morris, 1975). It is within this structure that the umbilical artery and vein are formed and through which the exchange of material between embryonic and maternal circulation takes place. Without this connection to the maternal circulation, embryonic nutritional resources and oxygen are quickly exhausted resulting in stunted embryonic growth and fetal death by 10.5 dpc. Normally, the allantois has fused to the chorion by 9.5 dpc (Rugh, 1990) as shown in Fig. 5. The allantois can be seen as a fine structure extending continuously from the gut of the embryo to the developing placenta. Initial formation of blood vessels can also be seen within the allantois. The allantois was radically different in approximately 50% of the VCAM-1 −/− embryos (9 out of 17, 9.5 dpc −/− embryos examined in detail). The allantois was usually present as a large, swollen sac, which was not connected to the chorion (Fig. 5C-F). Normally, at this stage, it is impossible to separate the chorion and allantois without tearing the structure; however, in −/− embryos showing signs of defective chorioallantois formation, the free allantois could be easily identified prior to removal of the extraembryonic membranes (Fig. 5C), and, after removal, the embryo could be readily separated from the placenta with the allantois intact (Fig. 5D,E) The embryo shown in Fig. 6F contained an allantois that also had failed to fuse to the chorion but was not swollen, being present as a small free structure. Two embryos, like the one shown in Fig. 5G, were also found in which the allantois appeared relatively normal, but had fused to the placenta on its side, rather than on the very distal end that normally expresses VCAM-1. Within this type of allantois no vessels could be seen that were continuous with the placenta, suggesting that mutants displaying this phenotype would not be viable. Taken together, the reciprocal pattern of expression of VCAM-1 and α4 in the allantois and the chorion, and the failure of these two structures to fuse in VCAM-1-deficient embryos, suggest that the two counter receptors are required for formation of the chorioallantois. The failure to form the chorioallantois could, therefore, account for the death of VCAM-1 −/− embryos before 11.5 dpc. The failure of the allantois to fuse with the chorion does not, however, necessarily account for the death of embryos surviving beyond this stage. This is suggested by the observation that not all −/− embryos were affected at 9.5 dpc. Only 9 out of 17 homozygous −/− embryos examined in detail contained an abnormal allantois. The other 8 embryos had a fused chorioallantois that was normal in appearance (data not shown). Further, a detailed examination of the 11.5 dpc embryos showed that all surviving homozygous −/− embryos that were grossly normal in appearance had developed an umbilical artery and vein that was firmly attached to the placenta. The appearance of 11.5 dpc WT, heterozygous and VCAM-1 −/− embryos is documented in Fig. 6. VCAM-1-deficient embryos at this stage were viable (as judged by normal gross morphology and the presence of a beating heart), or had died and were highly delayed or necrotic as shown in Fig. 6C. Such embryos lacked a visible allantois or umbilical vessels, consistent with the notion that death occurred due to the failure to develop a fused chorioallantois. The majority of VCAM-1 −/− embryos viable at 11.5 dpc appeared as shown in Fig. 6D. The umbilical artery and veins were present, continuous with the placental vasculature and
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filled with blood, indicating that they were functional. Thus, in at least half of the VCAM-1 −/− embryos, placentation and development of the umbilical circulation appeared to have progressed sufficiently to support growth of the embryo until 11.5 dpc and, in some cases to 12.5 dpc. Multiple abnormalities in the hearts of VCAM-Ideficient embryos Although VCAM −/− embryos surviving until 11.5 dpc were normal in appearance, most embryos failed to survive beyond 12.5 dpc and none survived up to 13.5 dpc. The distribution of VCAM-1 protein at this time suggests that VCAM-1 may play some role in heart development that might account for failure to develop past 12.5 dpc. Normally, by 11.5-12.5 dpc, development has advanced to the point that the heart is four chambered, beating productively, and has clearly delineated ventricles and atria. Valve formation is well underway and the endocardial cushions are well developed. Septation of the outflow tract into the aorta and pulmonary artery has commenced, the epicardium has completely encased the ventricles and most of the atria, and the formation of capillaries and coronary vessels has started. Comparable sections from paraffin embedded hearts of WT and VCAM-1 −/− 11.5 dpc litter mates were prepared and analyzed (Fig. 7). The 11.5 dpc VCAM-1 −/− embryos used in these analysis were, in general, normal in appearance upon macroscopic examination (eg Fig. 6D). They also had normal sized hearts with specific structural features such as atrial valve leaflets and septum primum (Fig. 7A,D). However, all homozygous null embryos had blood in the pericardial sac, although not in sufficient amounts to cause a general swelling in the pericardium (compare Fig. 7A with D and B with E). While the origin of the bleeding within the pericardial space is not known, one explanation may be a defect in or damage to small coronary blood vessels that form underneath the epicardium. In particular, the atrial ventricular sulcus (avs), contains rapidly proliferating endothelial cells and mesenchymal cells that will form such subepicardial vessels (Viragh and Challice, 1981). As shown in Fig. 7C, the avs from WT hearts contained a pocket of densely packed cells consistent with the description of those that will form cororary vessels. In contrast, this pocket of cells was absent from the avs of VCAM-1 −/− hearts. The hearts of all 11.5 dpc VCAM-1-deficient embryos examined also lacked an epicardium. At this stage the epicardium can readily be detected as a continuous cover overlying the ventricular and atrial myocardium of normal embryos. Interestingly, small numbers of epicardial cells were detected on the atrium of 11.5 dpc −/− embryos (Fig. 7F) while the adjoining ventricular myocardium appeared completely devoid of epicardial cells. Several abnormalities were also noted in the ventricular myocardium of VCAM-1-deficient embryos. While the myocardial mass of the atria of VCAM-1 −/− hearts was similar to WT embryos, there was a reduction in the size of the developing intraventricular septum as well as a variable reduction in the thickness of the ventricular myocardium. In addition, the ventricular myocardium of the VCAM-1-deficient hearts appeared less compact and the cells more randomly organized when compared to control hearts (compare Fig. 7B and E). Occasional blebs of myocardium were observed extruding from the myocardial walls (Fig. 7D).
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Fig. 6. Appearance of VCAM-1-deficient embryos isolated at 11.5 dpc. The genotypes of the embryos are shown in the upper right hand corner of the photographs. The embryos are all from the same litter. The locations of fusion of the umbilical arteries and veins with the placenta are indicated by arrowheads. The embryos shown in A, B and D were approximately the same size and were taken at slightly different magnifications in order to fill the frame for photographic purposes. The embyro shown in C was considerably smaller than its litter mates and was approximately the size of a 10.5 dpc embryo.
The alterations observed in the developing hearts at 11.5 dpc were more pronounced in hearts of mutant embryos that survived until 12.5 dpc (Fig. 8). There was a significantly larger amount of blood in the pericardial sac than at 11.5 dpc, although this pericardial effusion was, again, not associated with marked pericardial swelling. The 12.5 day −/− hearts also failed to develop a complete intraventricular septum, while the WT hearts clearly contained fully septated ventricles. Also, the compact zone of the ventricular myocardium in the −/− hearts was severely reduced in thickness and appeared only slightly more developed than at 11.5 dpc. Thus, the hearts of the VCAM-1 embryos displayed abnormalities consistent with the
normal pattern of VCAM-1 expression suggesting that VCAM-1 is required for normal cardiac development. DISCUSSION Although our knowledge of genetic regulatory mechanisms active in development is advancing rapidly, to date, little is known about molecules that regulate important cell/cell and cell/extracellular matrix interactions as they relate to specific morphogenic events. In this report, the potential developmental role(s) in embryogenesis of one cell adhesion molecule,
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Fig. 7. The hearts of VCAM-1-deficient embryos isolated at 11.5 dpc show multiple abnormalities. The hearts of a WT and VCAM-1 −/− embryo were examined histologically as described in the text. A, B, and C are micrographs of the same WT embryo. The micrographs shown in D, E and F are from the same VCAM-1 −/− embryo. Both embryos are littermates. The micrographs in A and D are at 80× magnification, all others are at 320× magnification. The following structures are indicated. Atrial valve leaflets (v), septum primum (p), endocardial cushions (c), intraventricular septum (s),ventricular grove (g) epicardium (e) and atrial ventricular sulcus (avs). The micrographs in B and E are the center of the ventricle at the base (ventricular groove) of the intraventricular septum. Note the large amount of blood below the ventricular groove within the pericardial space in E. The micrographs in C and F are of the atrial/ventricular junction and avs located immediately to the right of the endocardial cushions and immediately below the septum primum. Note the pocket of cells in the avs of the WT heart (indicated with the curved arrow) that is absent in the −/− heart. The rows of epicardial cells from WT hearts are indicated by horizontal arrowheads. The locations of scattered epicardial cells present on the atrial wall of the homozygote VCAM-1 −/− embryo are indicated by vertical arrowheads.
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Fig. 8. The hearts of VCAM-1-deficient embryos isolated at 12.5 dpc display multiple abnormalities including a reduction in the ventricular myocardium. Specific structures are as indicated for Fig. 7. A and B are the same WT embryo and C and D are the same VCAM-1 −/− embryo. The thick myocardial wall of the ventricular compact layer is indicated (m). The micrographs in A and C are at 70× magnification while those in B and D are 280× magnification.
VCAM-1, was investigated by ablation of the VCAM-1 gene through targeted homologous recombination. These experiments demonstrated that VCAM-1-deficient embryos failed to develop past 12.5 dpc, and that mutant embryos died at either of two times. One group died at or shortly after 10.5 dpc, while the other remained viable for another 1 to 2 days. It seems unlikely that these results were due to incomplete inactivation of the VCAM-1 gene because of the severe nature of the mutation introduced into the VCAM-1 locus, and the absence of detectable VCAM-1 protein −/− embryos. There was also no indication that the mutation was leaky, in that only one homozygous mutant animal out of 900 F1 progeny was viable and this animal died before its genotype could be confirmed. It is more likely that the biphasic pattern of death represents the effects of VCAM-1 deficiency on multiple, critical developmental processes. The embryonic lethality was surprising in that the only previously suggested developmental role for VCAM-1 was as a mediator of myotubule fusion during skeletal muscle formation, which would not be expected to be
limiting at these stages. Also, to our knowledge, other mouse strains, described so far, that are deficient in expression of cell adhesion molecules expressed principally by endothelial cells in adults, including all three selectins and ICAM-I, are viable (Labow et al., 1994; Mayadas et al., 1993; Sligh et al., 1993; Arbones et al., 1994). These data suggest that VCAM-1 evolved first as a mediator of early developmental processes and was later adapted for use by the immune system. Requirement for VCAM-1 during placentation Several observations demonstrate that a VCAM-1/α4 interaction is critical for efficient placentation. First, VCAM-1 expression in the early postimplantation embryo was detected in the distal end of the forming allantois, and α4 was expressed in the adjacent chorionic plate for 48 hours prior to formation of the fused chorioallantois. Second, the allantois failed to attach firmly to the chorion in approximately half of the VCAM-1 −/− embryos. Third, failure to form the chorioallantois was the first phenotype observed in the VCAM-I −/−
VCAM-1-deficient mice embryos and occurred before any deleterious effects on embryonic survival or morphology were noted. Fourth, embryos deficient in expression of the α4 subunit of integrins also failed to develop a chorioallantois, resulting in approximately the same frequency of embryonic death at this stage (Yang et al., 1995). Since effective communication between embryonic and maternal circulation is required for survival beyond 10 dpc (Steven and Morris 1975), the failed placentation of a proportion of mutant embryos due to the absence of normal VCAM-1/α4 integrin interaction likely accounts for embryonic death prior to 11.5 dpc. The mechanism by which VCAM-1 and α4 integrins facilitate the joining of the allantois and the chorion is unclear. The simplest model assumes a direct interaction between VCAM1 on the allantois and α4 integrins on the juxtaposed chorion that is required for fusion. Alternatively, VCAM-1 and α4 binding may function in initial recognition, perhaps assuring that the allantois adheres to the chorion and not to other regions of the extraembryonic membranes (as was observed in two VCAM-1 −/− embryos), or to stabilize the initial interaction prior to the establishment of stable structures such as tight junctions and desmosomes. In this case, the need for VCAMI/α4 adhesion may only be transient, functioning to align the two structures and to facilitate the initiation of a cascade of events that more firmly establish the chorioallantoic connection. This model is consistent with the fact that VCAM-1deficient embryos that survived until 11.5 dpc were grossly indistinguishable from control litter mates, displaying normally developed chorioallantoic plates and well formed umbilical arteries and veins. Thus, even though VCAM-I/α4 might promote efficient and timely alignment of the allantoic stalk and the chorionic membrane, their interaction may not be absolutely required for normal placentation providing the advancing allantois manages to contact the chorion during some ‘receptive’ window of placental development. Requirement for VCAM-1 in normal cardiac development Survival of nearly half the mutant embryos to between 11.5 and 12.5 dpc provided the opportunity to study the effect of VCAM-I deficiency on later embryonic development. The only consistent defects observed in the surviving VCAM-1 −/− embryos isolated at 11.5 dpc, prior to obvious necrosis, were in the developing hearts. The lack of VCAM-1 expression had no effect on the folding of the heart, the positioning of the atrial and ventricular chambers or the septation of the outflow tract. In addition, the endocardial cells of the mutant hearts appeared to form a continuous inner lining of the myocardium with no obvious difference in the relationship of the endocardial cells to the underlying myocardium. Similarly, the endocardial cells overlying the endocardial cushions became fibroblastic, ceased expressing PECAM-1, began to express α4 and invaded the endocardial cushions in much the same manner, and within the same time table, as noted in hearts from WT embryos. However, marked morphological differences between control and VCAM-1 hearts at these later stages of development were readily apparent upon histological examination. Blood cells were always found within the pericardial sac of hearts from VCAM-1 −/− embryos, suggesting that the integrity of the vascular walls was compromised. Perhaps related to this, is the observation that the epicardial covering
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was absent from the ventricles and, if present at all, only as small patches of cells in the vicinity of the atrium. This observation is consistent with VCAM-1 and α4 integrins functioning to stabilize the adhesion of the epicardial layer to the myocardium. A direct role for VCAM-1 in the stable adherence of epicardial cells is supported by the observations that α4 integrins were expressed by the single layer of epicardial cells covering hearts from WT embryos, and that VCAM1 was expressed in a complementary fashion by cells of the underlying compact myocardial layer. Thus, it seems likely that the absence of the epicardium is directly due to loss of VCAM-1. It is relevant to note that the epicardium develops by invasion of the heart with mesothelial cells derived from the septum transversum that migrate over the myocardium to form the epicardial covering (Viragh and Challice, 1981). The migration of the epicardial precursors across the myocardium begins at around 9 dpc, at which time VCAM-1 is already expressed throughout the myocardium. The role of VCAM-1 and α4 integrins in the stable adhesion of epicardial cells to the myocardium would be directly analogous to the role of the counter receptors in the stable adhesion of migrating leukocytes to the endothelium. Although the physiologic consequences of the loss of the epicardium are unknown, the epicardial layer might play a functional role in heart development such that its absence could contribute to the phenotype of the VCAM-1 mutants. Several reports have correlated in time the formation of coronary vessels and the establishment of the epicardium. The coronary vessels appear to form in the space between the epicardium and the myocardium and are thought to be the result of endocardial precursors migrating and proliferating beneath the epicardial layer (Viragh and Challice, 1981; Mikawa et al., 1992; Poelmann et al., 1993). The formation of the coronary vessels may require either direct contact with the epicardium or factors expressed by the epicardial cells. Thus, the loss of the epicardium might indirectly prevent the formation of subepicardial blood vessels. These vessels are of particular importance as they function to connect the developing myocardial capillaries to the coronary arteries. Subsequent bleeding could result from drainage of blood, normally destined for these vessels into the pericardium. It is also possible that the expanding coronary vasculature is sufficiently delicate that it cannot withstand the physical stress resulting from the beating embryonic heart or the fluid pressure required to maintain the embryonic circulation. An overlying layer of epicardial cells might normally provide sufficient support to maintain the integrity of the early coronary vessels. Thus, seepage of blood into the pericardial space could result from physically damaged vessels. Consistent with both models is the observation that proliferating cells within the avs, thought to give rise to subepicardial blood vessels, were absent from VCAM-1 −/− hearts at 11.5 dpc. Also, subepicardial blood vessels were absent from hearts of 12.5 dpc VCAM-1 −/− embryos (data not shown). The phenotypes described above were remarkably similar to that observed for α4-deficient embryos (Yang et al., 1995). α4deficient embryos also had hearts that lacked an epicardium and that contained blood in the pericardial space. In addition, the subepicardial blood vessels were also missing in 12.5 dpc α4-deficient embryos. These observations strongly suggest that a VCAM-1/α4 interaction is critical for these developmental processes.
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An additional morphogenic defect consistently occurring in the hearts of VCAM-1 −/− mice involved the ventricular myocardium. The myocardial cells within the ventricle wall are normally present as a compact layer of organized, dividing cells that are the source of new cells for the expanding myocardial wall, the forming trabeculae and the intraventricular septum. Often the septum of VCAM-1-deficient embryos consisted of myocardial cells organized into a column that failed to extend to the ventricular roof and therefore could not establish the complete wall of myocardial cells required for the partitioning of the ventricle into two chambers. In severe cases, the septum was represented only by a mound of myocardial cells on the floor of the ventricle (Fig. 7D). The thick compact layer of myocardial cells that is several cells thick in the normal 12.5 day heart, was also thinner and less compact in appearance in the −/− embryos. The reduced thickness and loose organization of the ventricular myocardium, although variable in degree at 11.5 dpc, was consistently observed in hearts of embryos at 12.5 dpc, suggesting that VCAM-1 may be important in the initial alignment of cells prior to the establishment of a stable tissue structure or to the normal growth of the compact layer. Interestingly, the expression of VCAM-1 in much of the myocardium of the normal heart was observed in the absence of α4 expression. VCAM-1 was expressed in the earliest myocardial cells of the developing heart, prior to formation of the primordial heart tube (Baldwin et al., 1991), while α4 was not observed in the heart until expansion of the epicardium. Further, VCAM-1 was expressed in the intraventricular septum without expression of α4 in adjacent cells. These data, in conjunction with the observations that the myocardium is affected in VCAM-1-deficient mice but not in α4-deficient mice, suggest that VCAM-1 may have functions in the heart, independent of its interaction with the α4-expressing epicardium, that involve an as yet unidentified ligand. The ultimate death of embryos surviving beyond 10.5 dpc could be explained by one or a combination of the deficiencies noted here. The embryos may die due to abnormal cardiac function which may be attributed directly to the poorly developed myocardial walls as well as the lack of a correctly placed ventricular septum. The defect in the myocardium, in turn, may be due to the loss of VCAM-1 function or may be secondarily due to failure of the development of the epicaridium or coronary vasculature. It is also possible, as concluded from the analysis of independently derived VCAM-1-deficient mice (Gurtner et al., 1995), that many or all post-placentation defects observed in VCAM-1deficient embryos are due to compromised maternal/fetal interactions. However, the number of 11.5 dpc embryos, with apparently normal umbilical arteries and veins, observed here and in α4-deficient embryos suggests that the placentation defect is not the only factor determining the death of the embryos. In conclusion, the results presented here and by others (Yang et al., Gurtner et al., 1995) reveal new and unsuspected functions for adhesion molecules in embryonic development. These observations may have important implications in the etiology of congenital birth defects, provide new insights into problems of early fetal death and suggest caution in the use of anti-inflammatory agents directed against VCAM-1 or α4 during pregnancy.
The authors are grateful to Richard Hynes and Myron Cybulsky for helpful discussions and for sharing their observations on α4 and independently derived VCAM-1-deficient mice prior to publication. The authors thank Gwendolyn Wong and Jeanne Magram for helpful discussions. We also thank John Duker, Joseph Levine and Doug Larrigan for oligonucleotide synthesis and DNA sequencing. We thank Robert Terry and Joan Maccari for technical assistance. This work was supported by grants HL39023, HL47670, HL2917, CA10815, CA19144 and a Mary A. Smith lead Trust grant to H. S. B.
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