Volume 240, Issue 2 p. 384-393
Research Article
Free Access

The chemokine receptor CXCR7 functions to regulate cardiac valve remodeling

Sangho Yu

Sangho Yu

Gladstone Institute of Cardiovascular Disease, San Francisco, California

Department of Biochemistry & Biophysics, University of California, San Francisco, California

Department of Pediatrics, University of California, San Francisco, California

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Dianna Crawford

Dianna Crawford

Center for Immunology, Department of Medicine, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota

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Takatoshi Tsuchihashi

Takatoshi Tsuchihashi

Gladstone Institute of Cardiovascular Disease, San Francisco, California

Department of Biochemistry & Biophysics, University of California, San Francisco, California

Department of Pediatrics, University of California, San Francisco, California

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Timothy W. Behrens

Timothy W. Behrens

Center for Immunology, Department of Medicine, University of Minnesota Medical School, University of Minnesota, Minneapolis, Minnesota

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Deepak Srivastava

Corresponding Author

Deepak Srivastava

Gladstone Institute of Cardiovascular Disease, San Francisco, California

Department of Biochemistry & Biophysics, University of California, San Francisco, California

Department of Pediatrics, University of California, San Francisco, California

Gladstone Institute of Cardiovascular Disease, 1650 Owens Street, San Francisco, CA 94158Search for more papers by this author
First published: 18 January 2011
Citations: 62

Abstract

CXCR7 (RDC1), a G-protein-coupled receptor with conserved motifs characteristic of chemokine receptors, is enriched in endocardial and cushion mesenchymal cells in developing hearts, but its function is unclear. Cxcr7 germline deletion resulted in perinatal lethality with complete penetrance. Mutant embryos exhibited aortic and pulmonary valve stenosis due to semilunar valve thickening, with occasional ventricular septal defects. Semilunar valve mesenchymal cell proliferation increased in mutants from embryonic day 14 onward, but the cell death rate remained unchanged. Cxcr7 mutant valves had increased levels of phosphorylated Smad1/5/8, indicating increased BMP signaling, which may partly explain the thickened valve leaflets. The hyperproliferative phenotype appeared to involve Cxcr7 function in endocardial cells and their mesenchymal derivatives, as Tie2-Cre Cxcr7flox/− mice had semilunar valve stenosis. Thus, CXCR7 is involved in semilunar valve development, possibly by regulating BMP signaling, and may contribute to aortic and pulmonary valve stenosis. Developmental Dynamics 240:384–393, 2011. © 2011 Wiley-Liss, Inc.

INTRODUCTION

CXCR7 (RDC1) is an evolutionarily conserved, seven-transmembrane G-protein-coupled receptor (GPCR) with motifs characteristic of chemokine receptors, such as the DRY motif. It is expressed in brain, heart, kidney, spleen, thymus, and various tumors and associated vascular endothelial cells (Heesen et al.,1998; Madden et al.,2004; Burns et al.,2006; Miao et al.,2007). CXCR7 binds to CXCL12 (SDF-1) with high affinity (Balabanian et al.,2005; Burns et al.,2006), challenging the notion that CXCL12 has a monogamous relationship with CXCR4, as suggested by the nearly identical phenotypes of CXCL12 and CXCR4 knockout mice (Nagasawa et al.,1996; Ma et al.,1998; Tachibana et al.,1998; Zou et al.,1998). CXCR7 also binds to another chemokine, CXCL11 (I-TAC) (Burns et al.,2006). However, the significance of ligand binding to CXCR7 is unclear, since CXCR7 does not appear to transduce any intracellular signaling, such as Ca2+ mobilization or mitogen-activated protein kinase (MAPK) signaling, which are the hallmarks of chemokine receptor activation (Burns et al.,2006; Proost et al.,2007; Boldajipour et al.,2008; Hartmann et al.,2008).

Despite the lack of ligand-mediated signaling, expression of and ligand binding to CXCR7 has significant cellular and physiological consequences. During CXCR4-mediated migration of primordial germ cells (Boldajipour et al.,2008) and lateral line primordium (Dambly-Chaudiere et al.,2007; Valentin et al.,2007) in zebrafish, CXCR7 creates an essential CXCL12 gradient by sequestering and internalizing CXCL12. CXCR7 forms a heterodimer with CXCR4 and modulates certain aspects of its function (Sierro et al.,2007; Hartmann et al.,2008; Kalatskaya et al.,2009; Levoye et al.,2009). CXCR7 is also important in CXCR4-mediated transendothelial migration of tumor cells, but not in intra-tissue chemotaxis (Zabel et al.,2009). Overall, CXCR7 is an atypical chemokine receptor that does not directly activate downstream pathways, but rather modifies or fine-tunes the activation of CXCR4 in response to CXCL12. In certain environments, CXCR7 may also have CXCR4-independent functions, as it promotes cell survival and adhesion even without ligand binding; the mechanism is unknown (Raggo et al.,2005; Burns et al.,2006; Miao et al.,2007).

Cardiac valve morphogenesis is a complicated and delicate process involving myriad signaling events (Armstrong and Bischoff,2004). We found that CXCR7 is expressed in developing and adult heart, especially in endocardial cells and their mesenchymal derivatives. Given the complementary expression pattern of CXCR4 and CXCL12 in the heart and their identical loss-of-function cardiac phenotypes in mice, we speculated that CXCR7 is important for cardiac development. The consequences of CXCR7 deletion have been reported by two groups, but the phenotypes have been divergent, leaving its function in vivo unclear, particularly regarding CXCR7's effects on cardiac development.

In this study, we generated Cxcr7 floxed mice and characterized the global and cell-specific consequences of complete loss of Cxcr7 function. Complete deletion of Cxcr7 produced perinatal lethality due to enlarged semilunar (aortic and pulmonary) valves and occasional ventricular septal defects (VSDs). Endothelial-specific deletion of Cxcr7 mediated by Tie2-Cre caused a similar, but less severe, cardiac phenotype. The valve defects were due to increased proliferation of valve mesenchymal cells associated with increased bone morphogenetic protein (BMP) signaling. Our results suggest that CXCR7 is required for proper cardiac semilunar valve morphogenesis and normal BMP signaling.

RESULTS

Expression Pattern of CXCR7 in Mice

To examine the expression pattern of CXCR7 in the embryonic heart, we performed whole-mount in situ hybridization. In E10.5 mouse embryos, Cxcr7 was expressed at high levels in the prosencephalon, especially in the nasal process, and in a part of the rhomboencephalon and at lower levels in the neural tube, somites, and heart (Fig. 1A). A similar pattern was observed in postnatal day 1 (P1) mice (Fig. 1B). In E10.5 heart, Cxcr7 was mainly expressed in endocardial cells and their endocardial cushion mesenchymal cell derivatives, in both the outflow tract (OFT) and atrioventricular canal (AVC) regions (Fig. 1C–F) and to a lesser degree in myocardial cells (Fig. 1C,E). A portion of OFT cushion mesenchymal cells are also derived from cardiac neural crest cells and they also seemed to express Cxcr7 since all OFT cushion cells were Cxcr7+, even though neural crest cells did not express Cxcr7.

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Expression pattern of Cxcr7 mRNA in developing mouse heart. A: Whole-mount in situ hybridization was carried out with digoxigenin (DIG)-labeled Cxcr7 riboprobe at E10.5. Cxcr7 mRNA is expressed in the prosencephalon, rhombencephalon, somites, neural tube, and heart. h, heart; nt, neural tube; pro, prosencephalon; rh, rhomboencephalon; so, somite. B: Section in situ hybridization image of sagittally sectioned P1 mouse. Cxcr7 is expressed in various tissues, including brain, thymus, heart, stomach, and liver. Adr, adrenal gland; Br, brain; Cb, cerebellum; H, heart; Li, liver; Lu, lung; M, muscle; ONE, optic nerve ending; PU, penile urethra; R, rectum; Sk, skin; St, stomach; T, tooth; Th, thymus; Vb, vertebrae. C–F: Transverse sections made after whole-mount in situ hybridization. Cxcr7 is expressed in endocardial cells and endocardial cushion mesenchymal cells in both outflow tract (OFT) and atrioventricular canal (AVC) regions. Its expression in myocardial cells is much weaker than that of endocardial cells. D and F are high-magnification images of OFT and AVC endocardial cushion regions, respectively. En, endocardium; ECC, endocardial cushion; Myo, myocardium; A, atrium; V, ventricle; RV, right ventricle.

Perinatal Lethality of Cxcr7-Deficient Mice

To test the function of CXCR7 in mice, we generated mice with loxP sites flanking the Cxcr7 gene (Cxcr7flox/flox) and used them to create ubiquitous or endothelial cell-specific Cxcr7 knockout mice. A targeting vector was generated to insert loxP sequences immediately upstream and downstream of Cxcr7 exon 2 by homologous recombination with the Cxcr7 locus (Fig. 2A). Removal of exon 2, which contains the entire coding region of Cxcr7, by Cre-mediated recombination would result in the complete loss of CXCR7 function. After confirmation by Southern blot, correctly targeted embryonic stem cell clones (Fig. 2B) were injected into blastocysts. Chimeric offspring were crossed with wild-type C57BL/6 mice to generate Cxcr7flox mice, which were bred with deleter transgenic mice (Schwenk et al.,1995) to allow Cre-mediated recombination in all cells. F1 mice were out-crossed with wild-type C57BL/6 mice to establish a stably transmitting mouse line with heterozygous deletion of Cxcr7 exon 2 (Cxcr7+/−) (Fig. 2C).

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Generation of targeted Cxcr7-null mice. A: Schematic representation of the wild-type Cxcr7 locus, the targeted allele, and the deleted locus. Shaded regions represent exon 2 (exon 2, top); the cloning fragment that includes exon 2 and about 100 bp up- and downstream of exon 2 (Ex2, middle); the 5′ homologous arm, which was cloned from DNA immediately upstream of Ex2 (5′ arm, middle and lower); and the 3′ homologous arm, which was cloned from DNA immediately downstream of Ex2 (3′ arm, middle and lower). Black triangles indicate LoxP sites; arrowhead indicates primer sites used for genotyping. E, EcoRV; neo, neomycin-resistance gene cassette. B: Southern blot analysis of ES cell clones showing non-targeted (+/+) and targeted (+/flox) clones. The 9.4-kb wild-type and 7.1-kb targeted allele fragments digested with EcoRV (E) were identified with an upstream probe, as shown in A. C: PCR analysis of genomic DNA from E16.5 wild-type (+/+) embryos or embryos heterozygous (+/−) or homozygous (−/−) for Cre-mediated deletion of the targeted locus. The 3.9-kb wild-type and 1.9-kb deleted products were from a single set of primers outside of exon 2, as shown in A. D: Genotypes of offspring from Cxcr7+/− × Cxcr7+/− crosses at weaning and at E16.5. E: Six of eight E18.5 mice from a single (Cxcr7+/− × Cxcr7+/−) litter, photographed less than 1 hr after cesarean birth. Identifier and genotypes are shown for each mouse. F,G: Frontal views of E18.5 wild-type and Cxcr7−/− hearts. In some Cxcr7−/− hearts (22%), the aorta was misaligned and displaced to the right side (arrow). Ao, aorta; PA, pulmonary artery; ra, right atrium; la, left atrium; rv, right ventricle.

When Cxcr7+/− mice were inter-crossed, no Cxcr7−/−mice were found at weaning (Fig. 2D). A normal Mendelian ratio was observed at E16.5 (Fig. 2D) without abnormalities in size, color, or gross anatomy. About a third of E18.5 Cxcr7−/− mice delivered by cesarean birth at E18 were dead. The remaining mice died shortly after birth; most were cyanotic and gasped for breath, but the phenotype was variable. For example, in a litter of eight mice, one of three Cxcr7−/− mice died at or just before birth and appeared to have a catastrophic circulatory failure in late development, as it was completely white within a blood-filled embryonic sac (mouse 18-12, Fig. 2E). The second appeared anatomically normal but never breathed (mouse 18-10, Fig. 2E). And the third gasped and remained cyanotic before dying within 1 hr after birth (mouse 18-6, Fig. 2E). Some Cxcr7−/− mice appeared normal and robust at birth but then showed signs of stress, such as gasping for breath, and died suddenly.

CXCR7 Deficiency Causes Thickening of Semilunar Valves and Ventricular Septal Defect

To analyze the phenotypes of Cxcr7−/− mice in detail, we harvested E18.5 embryos from intercrosses of Cxcr7+/− mice, and examined the internal organs. In Cxcr7−/− embryos, the lungs appeared normal and were able to inflate. All other internal organs looked grossly normal except the hearts, which were slightly larger than age-matched wild-type hearts and occasionally had dilated atria or malalignment of the aorta (Fig. 2F,G).

Transverse and coronal sections of E18.5 Cxcr7−/− hearts revealed thickened semilunar valves (pulmonary and aortic valves) in all embryos; atrioventricular (tricuspid and mitral) valves were normal (Fig. 3A–J). One third of the embryos (n=9) had VSDs in the membranous portion of the septum, and 22% had overriding aortas (Fig. 3I). In contrast to a previous report (Sierro et al.,2007), we did not observe atrial septal defects or bicuspid semilunar valves. The perinatal lethality most likely resulted from insufficient blood flow to the body caused by semilunar valve stenosis, a condition that can cause severe cardiac dysfunction in human newborns as well.

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Cxcr7−/− embryos have enlarged semilunar valves and occasional VSD or overriding aorta. A–J: H&E-stained sections of E18.5 embryos. Cxcr7−/− embryos have thick pulmonary valve (PV) and aortic valves (AoV) but normal tricuspid and mitral valves. VSD was observed in 33% (n=9) of cases (arrow in I). Transverse sections are shown in A, C, F, and H. Coronal sections are shown in B, D, E, G, I, and J. Ao, aorta; PV, pulmonary valve; RV, right ventricle; LV, left ventricle; LA, left atrium; RA, right atrium; MV, mitral valve; TV, tricuspid valve. K–R: Coronal sections of hearts from wild-type and Cxcr7−/− embryos at various developmental stages. The sizes and cell numbers of OFT endocardial cushions or semilunar valves were not significantly different in wild-type and Cxcr7−/− embryos until E14. Difference in the size of semilunar valves became apparent at around E15.5, when the valves undergo a remodeling process and form thin leaflets in wild-type hearts, but remained enlarged in Cxcr7−/− hearts.

To determine when the semilunar valve thickening occurred during development, heart sections were made at progressive mouse embryonic stages. Until around E14.0, there was no apparent difference in the size of the OFT endocardial cushions (Fig. 3K–M and O–Q). However, at E15.5, when wild-type valve thinning into leaflets had already begun, Cxcr7−/− pulmonary and aortic valves were still thick, indicating defects in semilunar valve remodeling (Fig. 3N,R). Mitral and tricuspid valves were normal in mutants. Since OFT endocardial cushion is composed of both endocardial- and neural crest–derived cells, while AVC endocardial cushion is almost exclusively derived from endocardial cells (Hutson and Kirby,2007; Snarr et al.,2008), this result may reflect function of Cxcr7 in both cell types.

Increased Cell Proliferation in Semilunar Valves of Cxcr7−/− Embryos

Some neural crest cells originating at the level of the posterior rhombencephalon migrate to the heart through pharyngeal arches 3, 4, and 6 and contribute to various developmental events, such as outflow septation, great vessel alignment, and remodeling of pharyngeal arch arteries (Hutson and Kirby,2003; Stoller and Epstein,2005). Since valve thickening was observed only in OFT valves, and VSD and overriding aorta were present in some cases, we speculated that CXCR7 is involved in the migration of cardiac neural crest cells. However, whole-mount in situ hybridization for Crabp1 mRNA, a marker of neural crest cells, at E10.5 did not reveal any gross abnormalities between neural crest migration to pharyngeal arches 3, 4, and 6 in wild-type and Cxcr7−/− embryos (data not shown).

To investigate whether the proliferation or apoptosis of semilunar valve mesenchymal cells was affected in Cxcr7−/− embryos at various developmental stages, we analyzed cell proliferation by phospho-histone H3 (pH3) immunofluorescence (Fig. 4A,B) and cell death by TUNEL assay. In wild-type semilunar valves, the percentage of cells undergoing mitosis decreased as embryos grew older. In contrast, Cxcr7−/− valves had ∼2-fold more pH3-positive semilunar valve cells than wild-type valves at E14.0 and 3.5-fold more at E15.5 (Fig. 4C). No differences were detected in the percentage of pH3-positive cells in pulmonary valves compared to aortic valves. The percentages of cells undergoing apoptosis were almost identical in wild-type and Cxcr7−/− valves (data not shown).

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Increased proliferation of cardiac semilunar valve mesenchymal cells in Cxcr7 mutants. A,B: Cells undergoing mitosis were stained with pH3 antibody (red) at various embryonic stages. Nuclei were counterstained with DAPI (blue). There were more proliferating cells in semilunar valves in Cxcr7−/− embryos from E14 onward. Shown are representative sections at E15.5. C: Quantitative analysis of pH3 immunostaining (pH3-positive cells/total valve cells × 100) showed that the percentage of cells undergoing mitosis in Cxcr7−/− valves was increased ∼2-fold at E14 and ∼3.5-fold at E15.5. *P < 0.01.

CXCR7 in Endocardial Cells Is Important for Semilunar Valve Morphogenesis

To identify the cell type in which CXCR7 functions during valve development and to examine the role of CXCR7 in the adult heart, we deleted Cxcr7 specifically in endothelial and endocardial cells using the Tie2-Cre transgene. Tie2-CreCxcr7flox/− mice survived normally after birth and were fertile. However, at 6 months of age, they had marked cardiac hypertrophy (Fig. 5A). Cross-sections of age-matched wild-type and Tie2-CreCxcr7flox/− hearts revealed much thicker ventricular walls in mutant hearts, but no difference in the size of the ventricular cavity (Fig. 5B).

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Endothelial-specific deletion of Cxcr7 causes cardiac semilunar valve stenosis and secondary hypertrophy. A: Image of whole hearts taken from 6-month-old wild-type or Tie2-CreCxcr7flox/− mice. Endothelial-specific deletion of Cxcr7 caused marked hypertrophy. B: Transverse sections of wild-type and Tie2-CreCxcr7flox/− mice. The mutant heart had a thicker ventricular wall than the wild-type heart but a similarly sized ventricular cavity. C: Left ventricular posterior wall (LVPW) thickness, measured by echocardiography, was ∼1.4-fold thicker in Tie2-CreCxcr7flox/− hearts than in wild-type hearts (n=3 per group, *P<0.05). D–G: Heart sections of 6-month-old wild-type or Tie2-CreCxcr7flox/− mice were stained with hematoxylin-eosin. Tie2-CreCxcr7flox/− mice had aortic valve (AoV) stenosis similar to that in Cxcr7−/− mice but a milder pulmonary valve (PV) phenotype. H,I: Magnified images (20×) of wild-type and Tie2-CreCxcr7flox/− heart sections stained with hematoxylin-eosin. Myocardial structure was disarrayed and the myocytes were not well packed in mutant hearts. J,K: Masson's trichrome staining showed more collagen deposition (blue) in Tie2-CreCxcr7flox/− hearts than in wild-type hearts.

Echocardiographic analysis of cardiac function at 6 months of age showed no difference in fractional shortening or ejection fraction between Tie2-CreCxcr7flox/− and wild-type mice (data not shown), but the left ventricular posterior wall was about 1.4-fold thicker in Tie2-CreCxcr7flox/− mice (Fig. 5C). These features are consistent with concentric hypertrophy caused by pressure overload (Jessup and Brozena,2003).

To determine whether Tie2-CreCxcr7flox/− mice had semilunar valve stenosis, we examined histological sections at the level of the semilunar valves. Indeed, the semilunar valves of Tie2-CreCxcr7flox/− mice were thicker than those of wild-type control mice, and the difference was more dramatic in the aortic valves compared to the pulmonary valves (Fig. 5D–G). This result suggests that Cxcr7 expression in endocardial cells and their valve mesenchymal derivatives is necessary to prevent thickening of the semilunar valves.

Examination of the myocardial structure of Tie2-CreCxcr7flox/− hearts at high magnification revealed greatly enlarged cardiomyocytes, cellular disarray, and increased intercellular space (Fig. 5H,I). At 6 months of age, the mutant mice had substantial fibrosis, marked by Masson's trichrome staining, present in the myocardium and around microvessels in the heart; little fibrosis was detected in wild-type mice (Fig. 5J,K).

BMP Signaling Is Enhanced in Semilunar Valves of Cxcr7 Mutants

A hypomorphic allele of epidermal growth factor receptor (EGFR) also results in thickening of semilunar valves (Chen et al.,2000), and mutation of one of its ligands, heparin-binding epidermal growth factor (HB-EGF), led to dysregulation of BMP signaling and similar valve stenosis in mice (Jackson et al.,2003). To determine whether BMP signaling was increased in Cxcr7−/− semilunar valves, we performed immunostaining for phospho-Smad1/5/8 (pSmad1/5/8), which transduces BMP signaling. pSmad1/5/8 levels were about 1.5-fold higher in Cxcr7−/− semilunar valves than in wild-type valves throughout development (Fig. 6A–E).

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CXCR7 deficiency enhances BMP signaling in semilunar valves. A–D: Immunohistochemistry with anti-phospho-Smad1/5/8 (pSmad1/5/8) antibody (brown) shows increased BMP signaling in Cxcr7−/− semilunar valves. Nuclei were counterstained with hematoxylin. E: Quantitation of pSmad1/5/8 immunostaining shows that the BMP signaling was increased throughout cardiac valve development in Cxcr7−/− mice. *P < 0.05.

DISCUSSION

This study shows that endothelial expression of the chemokine receptor CXCR7 is important in limiting proliferation of cells during cardiac semilunar valve morphogenesis. Cxcr7 germline deletion in mice caused perinatal lethality, likely because of insufficient blood supply to peripheral tissues as a result of severe aortic and pulmonary valve thickening and stenosis. Loss of CXCR7 also increased BMP signaling in semilunar valves and caused a sustained high proliferative rate of valve mesenchymal cells during the valve remodeling stage. Tie2-CreCxcr7flox/− mice with endothelial cell-specific ablation of Cxcr7 had similarly enlarged semilunar valves later in life and developed massive cardiac hypertrophy presumably due to aortic valve stenosis.

These results are consistent with the first report of a Cxcr7 mutant phenotype (Sierro et al.,2007), but not with a subsequent independent Cxcr7 knockout study (Gerrits et al.,2008). Perinatal lethality was observed in both studies. However, Gerrits et al. (2008) did not observe semilunar valve thickening or ventricular septal defects and concluded that the enlarged hearts in Cxcr7-null mice were due to hyperplasia. In contrast, Sierro et al. (2007) observed aortic and pulmonary valve thickening, as well as bicuspid aortic valves, a finding we did not observe. These discrepancies might reflect differences in genetic background, knockout strategies, or effects on neighboring genes. Gerrits et al. (2008) knocked in the β-galactosidase (LacZ) transgene in the place of Cxcr7 exon2, while we and Sierro et al. (2007) replaced the exon2 with a loxP-flanked conditional allele. No predicted microRNAs are in this locus, making it less likely that there are differences in the deletion of potentially embedded genes.

Semilunar valve thickening in Tie2-CreCxcr7flox/− mice confirms the endothelial origin of this phenotype. However, the less severe phenotype of Tie2-CreCxcr7flox/− mice compared to germline Cxcr7−/− mice suggests that Cxcr7 function in other cell types may also be important. Given the Cxcr7 expression in neural crest–derived cushion mesenchymal cells and lack of overriding aorta and VSD in Tie2-CreCxcr7flox/− mice, Cxcr7 may also have a critical function in neural crest–derived cushion cells during OFT maturation and septum formation. Alternatively, the imperfect recombination induced by Tie2-Cre might have prevented elicitation of the low penetrance VSD and overriding aorta (33 and 22%, respectively) phenotypes in Tie2-CreCxcr7flox/− mice. Further study is required to pinpoint the cellular origin of each phenotype and to elucidate the Cxcr7 function in neural crest–derived cells using neural crest–specific Cre mice.

Disruption of EGFR signaling pathways in mice also causes cardiac valve hypertrophy due to increased proliferation at the late stage of valve development (Chen et al.,2000; Jackson et al.,2003). There is evidence that EGFR signaling antagonizes BMP signaling (Kretzschmar et al.,1997; Nonaka et al.,1999; Lo et al.,2001), and pSmad1/5/8 levels may be increased in Hbegf knockout valves (Jackson et al.,2003), consistent with a link between EGFR signaling and Cxcr7. EGFR activation antagonizes BMP signaling by phosphorylating Smad proteins at the linker region distinct from phosphorylation sites by BMP receptor (Kretzschmar et al.,1997) or by stabilizing the Smad transcriptional co-repressor TGIF (Lo et al.,2001). Both effects are mediated by the MAPK Erk. In one study, Hbegf mRNA was downregulated in Cxcr7−/− semilunar valves, corroborating the relationship between EGFR signaling and CXCR7 (Sierro et al.,2007). It is unclear how CXCR7 regulates Hbegf expression. In our study, treating CXCL12 to primary sheep aortic valve mesenchymal cells, which express Cxcr7, or Cxcr7-transfected COS1 cells did not activate EGFR signaling (data not shown).

CXCR7 shares the same ligand, CXCL12, with CXCR4 but ligand binding to CXCR7 does not activate classical chemokine pathways such as Ca2+ mobilization or MAPK signaling (Burns et al.,2006; Proost et al.,2007; Boldajipour et al.,2008; Hartmann et al.,2008). Although CXCR7 can modulate the function of CXCR4 through ligand sequestration or heterodimerization (Dambly-Chaudiere et al.,2007; Sierro et al.,2007; Valentin et al.,2007; Boldajipour et al.,2008; Levoye et al.,2009; Zabel et al.,2009), semilunar valve thickening is a unique phenotype of Cxcr7 knockout mice that is not observed in either Cxcr4 or Cxcl12 knockout mice, where VSD is the major cardiac phenotype. In contrast, semilunar valve thickening is caused by both Cxcr7 deficiency and disruption of EGFR signaling, although VSD and overriding aorta are unique to Cxcr7−/− mice. Thus, CXCR7 may influence semilunar valve morphogenesis and ventricular septum formation/OFT maturation through two separate mechanisms. It will be interesting to test whether CXCR7 activates different pathways in endocardial- and neural crest–derived cushion mesenchymal cells.

Valve defects are among the most common forms of human congenital heart disease and typically involve abnormal thickening of the aortic or pulmonary valves. The consequences can range from neonatal cardiac failure to cardiac hypertrophy later in life, depending on the severity of the stenosis. Numerous signaling pathways have been linked to valve formation (Armstrong and Bischoff,2004). Disruption of any of these pathways during pregnancy could lead to fatal conditions, underscoring the importance of elucidating the exact mechanisms of valve formation. For example, the absence of Ptpn11, which is involved in a signaling pathway mediated by the EGFR, results in dysplastic outflow valves (Chen et al.,2000). In humans, mutations of PTPN11, which encodes the protein tyrosine phosphatase Shp-2, cause Noonan syndrome, characterized by pulmonic valve stenosis (Tartaglia et al.,2001). Similarly, human mutations in NOTCH1 (Garg et al., 2005) cause thickened aortic valve leaflets and can result in aortic valve stenosis early or later in life. It will be interesting to determine whether CXCR7 or pathways that it regulates also contribute to human aortic or pulmonary valve disease.

EXPERIMENTAL PROCEDURES

Generation of Conditionally Targeted Cxcr7 Mice

All experimental procedures were approved by the Institutional Animal Care and Use Committee at the University of California San Francisco. A conditional knockout targeting vector was designed to insert loxP sequences about 100–200 base pairs from either side of exon 2 of the Cxcr7 locus. The linearized targeting vector was electroporated into 129/SvEv embryonic stem (ES) cells (Dr. B. Koller, University of North Carolina, Chapel Hill, NC). ES clones were screened by PCR and verified by Southern blot. Correctly targeted ES cells were injected into C57BL/6 mouse blastocysts. Chimeric offspring were crossed with C57BL/6 mice, and agouti offspring were screened for the targeted allele, Cxcr7flox. Deleter Cre transgenic mice were used to allow Cre-mediated recombination in all cells (Schwenk et al.,1995). Tie2-CreCxcr7flox/− mice were generated by crosses of Cxcr7flox/flox × Tie2-CreCxcr7+/− or Cxcr7flox/− × Tie2-CreCxcr7flox/+. Tie2-CreCxcr7flox/+ mice were used as wild-type controls. Tie2-Cre mice (Koni et al.,2001) were purchased from the Jackson Laboratory (West Grove, PA).

In Situ Hybridization

Section in situ hybridization for P1 mouse was performed by Phylogeny (Columbus, OH). Mouse tissue was frozen, cut into 6–12μm sections, mounted on gelatin-coated slides, and fixed in 4% formaldehyde. The cRNA probe for Cxcr7 was synthesized in vitro according to the manufacturer's conditions (Ambion, Austin, TX) and labeled with 35S-UTP (Amersham, Pittsburgh, PA). Sections were hybridized overnight at 55°C with 35S-labeled cRNA probe (50–80,000 cpm/μl). Whole-mount in situ hybridization for Cxcr7 and Crabp mRNAs was performed with E10.5–E11.0 embryos (n=4 and 3, respectively) as described (Kwon et al.,2009). Embryos were fixed in 4% formaldehyde overnight and kept in 100% methanol at −20°C until use. Hybridization was performed at 65°C overnight. Riboprobes were labeled with DIG, and embryos were stained according to the manufacturer's instruction (Roche, Indianapolis, IN).

Histology

Embryonic hearts at various stages were embedded in paraffin, cut into 8–10-μm sections, deparaffinized, and stained with hematoxylin and eosin for histological analysis (n=4 for E11.5, n=3 for E12.5, n=3 for E14.0, n=3 for E15.5, and n=4 for E18.5). For Masson's trichrome staining, 6-month-old hearts from wild-type and Tie2-CreCxcr7flox/− mice were embedded in paraffin and transversely sectioned (n=3 for each group). Deparaffinized sections were dipped sequentially into Weigert's iron hematoxylin working solution for 10 min, Biebrich scarlet-acid fuchsin solution for 15 min, phosphomolybdic-phosphotungstic acid solution for 15 min, aniline blue solution 5–10 min, and 1% acetic acid solution for 2–5 min.

Immunostaining and TUNEL Assay

Paraffin-embedded embryos of wild-type and Cxcr7−/− mice were cut at various stages either transversely or coronally. For immunostaining, antibodies for phospho-histone H3 (Upstate, East Syracuse, NY) and phospho-Smad1/5/8 (Cell Signaling, Danvers, MA) were used at 1:100 dilution. For pH3 staining, a FITC-conjugated secondary antibody was used, and the slides were mounted with Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). For pSmad1/5/8 staining, a horseradish peroxidase–conjugated secondary antibody was used, and the sections were stained with the ABC staining system (Vector Laboratories). TUNEL assays were performed with an in situ cell death detection kit (Roche). Number of animals used: pH3 staining (n=3 for E11.5, E12.5, E13.5, and E14.0; n=5 for E15.5); pSmad1/5/8 staining (n=3 for E11.5 and E15.5, n=6 for E12.5 and E14.0); TUNEL assay (n=2 for E11.5, n=3 for E12.5, n=4 for E14.0 and E15.5). For wild-type and mutant mice, the same numbers of animals were used.

Echocardiography

Systolic function and ventricular wall thickness were assessed with M-mode and power Doppler mode in 6-month-old wild-type and Tie2-CreCxcr7flox/− mice (n=3 for each group); mice were anesthetized with 1.75% isoflurane. Core temperature was maintained at 37–38°C, and scans were performed in a random-blind fashion. Each mouse underwent three separate scans on three different days. Values were averaged from three scans.

Statistical Analysis

Statistical analysis was performed with the Student's t-test and P < 0.05 was considered statistically significant.