Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7

Abstract

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7(-/-) mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7(-/-) mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7(-/-) heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4.

Fig. 1.

Cxcr7^−/− mice develop a normal immune system. (A) CXCR7 and CXCR4 expression patterns in human resting leukocyte subsets. The color scale indicates transcript signal where black = absent, yellow = moderately expressed, and red = highly expressed. Cxcr7 mRNA levels quantitated by real-time PCR in human (B) and murine (C) T and B cells relative to naive or follicular (FO) B cells, respectively. Values ± SEM. (D) E17.5 fetal liver cells were stained with antibodies against B220 and CD43. Flow cytometry enumerations were performed within lymphocyte scatter gates. Percentages of cells in the marked gates are indicated. (E) Histological analysis of adult Cxcr7^−/− spleen. Sections were stained with antibodies against B220 (green) and CD1d (red). The splenic MZ is indicated (bar)..

Fig. 2.

SLV dysmorphogenesis in Cxcr7^−/− mice. Adult aortic valve from control; (A, C, and E) and Cxcr7^−/− (B, D, and F) mice. Arrows indicate leaflet boundaries. (A and B) Supravalvular view. Planes of sections shown in D and F are indicated. (C–F) Sections through the valves presented in A and B. (C and D) Safranin O staining; red indicates proteoglycans and glucosaminoglycans. (E and F) Movat pentachrome staining; blue indicates proteoglycans. Neonatal phenotype: (G and H) Dissected control and Cxcr7^−/− (−/−) hearts. Right ventricle (RV) is often dilated in Cxcr7^−/− mice. (I and J) Haematoxylin/eosin-counterstained sections of control and Cxcr7^−/− hearts. Membranous ventricular septal defects (VSD, red arrow) and sometimes overriding aorta can be seen in mutants. Dissected aortic (K–N) and Pulmonary (O–R) valves from control and Cxcr7^−/− neonates. Arrows indicate leaflet boundaries. Control aortic (K) and pulmonary (O) valves. (L–M, P–R): Phenotypical alterations in mutant valves ranging from thickening, with occasional fusion of valve leaflets (L and P), to formation of a bicuspid valve (M and Q), to obstruction of the valve by an overgrown leaflet (N and R). (S–V) Sections through the neonatal aortic (S and T) or pulmonary (U and V) valves of control (S and U) or Cxcr7^−/− (T and V) mice. Black arrows point to valve leaflets. AV, aortic valve; PV, pulmonary valve; LV, left ventricle; Ao, aorta.

Fig. 3.

Expression of Cxcr7, Cxcr4, and Cxcl12 during heart embryogenesis. ISHs with probes specific to Cxcr7 (A, C, E, and H), Cxcr4 (B, D, F, and I), and Cxcl12 (G and J). (A and B) Dissected E10.5 hearts. (C and D) Sections through endothelial cushions of A and B. (E–G) Sections throughout SLVs of E12.5 embryos. (H–J) Sections through myocardium of E14.5 embryos. Avc, atrioventricular canal; Oft; the ouflow tract points; Vm, valve mesenchyme. Arrowheads point to microvasculature; the arrow in H points to unstained coronary vessel.

Fig. 4.

Gene profiling of neonatal Cxcr7^−/− SLVs. Heat maps of ECM and adhesion genes differentially expressed in WT and Cxcr7^−/− dissected SLV leaflets (A), genes associated with SLV defects (B), and genes linked to Adm function (C). Signal intensity is color-coded (A–C). (D) qRT-PCR analysis of Hbegf and Adm expression in various control and Cxcr7^−/− neonatal organs. Values ± SEM. (E–H) PhosphoSmad1/5/8 or phosphohistone H3 immunohistological staining on sections of E14.5 control (+/+) and Cxcr7^−/− (−/−) SLVs. Percentage of positive nuclei in SLVs per staining and genotype are indicated. *, P < 0.01; **, P < 0.001.

Fig. 5.

CXCR4 and CXCR7 form functional heterodimers. (A) FRET measurements in unstimulated HEK293 cells transiently transfected with CXCR4-CFP and CXCR7-YFP. CFP emission before (CFPpre) and after YFP bleaching (CFPpost) and a false-color merged image (FRET) are shown. Data are expressed as percentage of FRET efficiency ± SD. (B) Representative Ca²⁺ flux triggered by CXCL12 on HEK293 cells expressing both CXCR7 and CXCR4 or only-CXCR4. Results are expressed as a percentage of the maximum chemokine-induced Ca²⁺ response. (C) Anti-phospho-ERK immunoblot after CXCL12 stimulation of HEK293 or CXCR7-transfected HEK293. Protein loading was controlled with an anti-ERK antibody.