Fig 2: Dynamic expression of CXCR4 and CXCL12 in the OFT and developing SLVs(A) Schematic representation of E10.5–E11.5 heart (sagittal view) showing endocardium (green), endocardial cushions (yellow), and distal-proximal axis of OFT. Sectors (a), (b), and (c) identify distal, medial, and proximal OFT cushion, respectively; transverse section from region (b) (medial OFT) is also depicted. Double-headed arrow indicates valve-forming region.(B–D’’) E11.5 wild-type OFT immunostained with anti-CXCR4 and PECAM-1 antibodies: distal region (a) in (B)–(B”); medial region (b) in (C)–(C”); and proximal region (c) in (D)–(D”). Boxed areas in (B)–(D) are shown enlarged in neighboring panels; double-headed arrows in (B) and (C) indicate valve primordia. Arrows in (B’), (B”), (C’), (C”), (D’), and (D”) show CXCR4+ ECs; yellow dashed lines indicate sub-endocardial CXCR4+ MCs, and white dashed lines indicate endocardium.(E) Bar graphs show relative distribution of CXCR4+ MCs (PECAM-1-) and ECs (PECAM-1+) within the OFT (valve-forming region and proximal OFT cushion) at E11.5.(F–G’) E12.5 (F–F’’) and E14.5 (G and G’) AoV, immunostained as above. Faint puncta in (F’) are shown enlarged in inset box; arrows in (F)–(F’’) indicate CXCR4+ ECs in SLV commissures.(H) Sagittal section of E11.5 wild-type OFT (immunostained as above). Boxed areas showing distal (i) and proximal (ii) OFT are enlarged in neighboring panels. Arrowheads indicate CXCR4+ MCs (PECAM-1-) in proximal cushion.(I) Graph shows proportion of CXCR4+ MCs with punctate CXCR4 in valve-forming and proximal cushion regions of E11.5 OFT (n = 4) and E12.5 SLVs (n = 4) as a percentage of total CXCR4+ MCs. Error bars represent ± SD; ***p = 0.001 (unpaired Student’s t test).(J–N) Cxcl12GFP/+ E11.5 OFT cushion (J–L) and E12.5 SLV (M and N) sections immunostained with anti-GFP, anti-CXCR4, and anti-PECAM-1 antibodies. Boxed region in (K) is shown enlarged in neighboring panels; arrowheads indicate GFP+/CXCR4+ cells.(O) Schematic showing relative levels of punctate or membranous CXCR4 (red) in MCs and ECs, and CXCL12 in the OFT wall and cushions (green) in distal, medial, and proximal OFT.Ant, anterior; EC, endocardial cell; MC, mesenchymal cell; NC, non-coronary; OFT, outflow tract; RA, right atrium; RV, right ventricle. Scale bars represent 100 µm. See also Figure S2.

Fig 3: Inhibition of PI3K affects orientation of CXCR4+ OFT mesenchymal cells in response to a CXCL12 gradient(A) Schematic representation of OFT explant assay using E10.5 wild-type embryos.(B) Sections of explants in collagen were immunostained with anti-CXCR4 (Cl. 247506) and anti-GM130 (Cl. EP892Y) antibodies; nuclei were stained with DAPI. Arrows indicate cellular orientation in relation to collagen surface (dashed line). Orientations were assigned according to sectors in the circular diagram.(C) Graph shows mean “away” orientated CXCR4+ MCs as a percentage of total CXCR4+ MCs after indicated treatment (minimum 3 explants per treatment group and >1,400 cells analyzed). Each treatment group mean was compared to the control group (serum-free only, black bar) using a one-way ANOVA test. Bars indicate mean ± SD. Comparisons achieving statistical significance are indicated by asterisks (**p = 0.01).endo, endocardium; myo, myocardium. Scale bars represent 25 µm.

Fig 4: Increased mesenchymal cell proliferation in Cxcl12-null SLVs and distinct SLV dysmorphogenesis following tissue-specific mutagenesis of Cxcr4(A–E) Timed knockout of CXCR4. Schematic (A) shows breeding scheme to generate Caggs-CreERT;Cxcr4fl/- embryos and timing of tamoxifen injection and analysis. Timed knockout mutant (C and E) and control (Caggs-CreERT;Cxcr4fl/+; B and D) SLV sections were immunostained with anti-CXCR4 and anti-PECAM-1 antibodies.(F–J) EdU incorporation in E12.5 and E15.5 SLVs. Wild-type and Cxcl12-/- AoV sections were immunostained with anti-CXCR4 and anti-PECAM-1 antibodies; red nuclei indicate incorporated EdU (F–I). Graphs in (J) compare EDU+ cells (as percentages of total CXCR4+ or CXCR4- MCs and total ECs) in Cxcl12 nulls and controls. Error bars represent ± SD; *p = 0.05, **p = 0.01, and ***p = 0.001 (unpaired Student’s t test).(K–R) 3D reconstructions (Imaris) of E15.5 SLVs from controls (K, L, O, and P), Tie2-Cre; Cxcr4fl/- mutants (M and N), and Pax3-Cre;Cxcr4fl/- mutants (Q and R). Leaflet color is as follows: green (right); blue (left); and red (non-coronary or anterior).(S) Graphs show total SLV volumes (mm3) for E15.5 Tie2-Cre;Cxcr4fl/- and Pax3-Cre;Cxcr4fl/- hearts (compared to Cxcr4fl/- littermate controls. Error bars represent ± SD; *p < 0.05 and **p < 0.01 (unpaired Student’s t test).(T–Y) Comparison of E12.5 control (Cxcr4fl/-; T and W) with Tie2-Cre;Cxcr4fl/- (U and X) and Pax3-Cre;Cxcr4fl/- (V and Y) mutant SLVs, whole-mount immunostained with anti-PECAM-1 antibody (confocal images).endo, endocardial; mes, mesenchymal. Scale bars represent 100 µm. See also Figure S4.

Fig 5: Confocal laser scanning micrographs of tCXCR4/CatCh protein in NG108-15 cells.a,b. Z-stack overlay of a typical tCXCR4/CatCh cell either treated with 50 nM SDF1α for 45 min (a) or with AMD3100 (b). The tandem construct is internalized by the action of SDF1α (similar to CXCR4 shown in Fig 2). c. Internalization of anti-CXCR4 antibody after immunoreaction with tCXCR4/CatCh in cells exposed to 50 nM SDF1α. The fluorescence signal of anti-CXCR4 (top, red) and tCXCR4/CatCh (middle, cyan) co-localize to a great extend as can be seen from white areas in the overlay (bottom). Scale bars represent 10 μm.