Effect of ackr3b knockdown on zebrafish embryo development. A-E Embryos injected with 0.2 pmoles/embryo of std-MO (A) or ackr3b-MO1 (B-E) were photographed at 28 hpf. Embryos with close-to-normal (B) and mild (C) phenotype were used for all further analyses whereas severe (D) and very severe (E) phenotypes were discarded. F, G Effect of ackr3b knockdown on neuromast migration. WISH analysis of claudin b expression was performed at 48 hpf on zebrafish embryos injected with std-MO (F) or ackr3b-MO1 (G) to assess actual ackr3b knockdown. Note the impaired neuromast migration in ackr3b morphants (arrows in G) when compared to the normal positioning observed in control embryos (arrows in F). The number of embryos presenting the showed phenotype in respect to the total number of analyzed embryos is shown in both panels.

WISH analysis of ackr3b and cxcr4a expression during zebrafish embryo development. WISH analysis of ackr3b(A–H) and cxcr4a(I–J) mRNA expression was performed at the indicated stages of development. (A–C,E,G,I,J) Lateral view of whole-mount embryos. (D) Coronal cross section through the trunk highlighted as a dashed vertical bar in (C), anterior to the top; arrows indicate somitic ackr3b expression. (F,H) Transverse cross sections through the trunk highlighted as dashed bars in (E,G), respectively. Arrows in (F,H) indicate ackr3b-positive developing gut and the basal part of the somites, respectively. (A–C,E,G,I,J) Scale bar: 100 μm. (D,F,H) Scale bar: 25 μm.

Effect of ackr3b knockdown on arterial/venous differentiation and lateral dorsal aorta (LDA) bifurcation. (A–F) WISH analysis of the arterial marker ephrin-B2a(A,B) and venous markers ephb4a(C,D) and flt4(E,F) expression was performed at 28 hpf on zebrafish embryos injected with std-MO (A,C,E) or ackr3b-MO1 (B,D,F). Arterial and venous marker expression is normal in ackr3b morphants when compared to controls. (G,H) WISH analysis of cadherin 5 expression was performed at 30 hpf on zebrafish embryos injected with std-MO (G) or ackr3b-MO1 (H) to investigate LDA formation. Note the interruption of LDA in ackr3b morphants [asterisk in H]. The number of embryos presenting the showed phenotype in respect to the total number of analyzed embryos is shown in each panel. Scale bar: 100 μm.

ackr3b knockdown affects ISV development and endothelial filopodia protrusions. Tg(kdrl:EGFP) embryos injected with std-MO (left panels) or ackr3b-MO1 (right panels) were photographed at 26 hpf (B,C) and 48 hpf (D,E) under an epifluorescence microscope. (B–E) High magnifications of the trunk region highlighted by the dotted box in (A) Asterisks in (C,E) indicate aberrant sprouts in ackr3b-MO1-injected embryos. Vasculogenesis occurs normally in ackr3b morphants as indicated by the presence of DA and PCV (brackets in B,C). (F–I) Confocal microscopy analysis of 28 hpf Tg(kdrl:EGFP) zebrafish embryos injected with std-MO (F,G) or ackr3b-MO1 (H,I). Note the aberrant filopodia protrusions from stalk cells in ackr3b morphants (arrows in I) when compared to control embryos. (G,I) High magnification of the region highlighted by dotted boxes in (F,H), respectively. (A–E) Scale bar: 100 μm. (F–H) Scale bar: 25 μm.

ackr3b knockdown impairs hlx1 expression. (A–D) WISH analysis of hlx1 expression in 26 hpf-embryos injected with std-MO (A,B) or ackr3b-MO1 (C,D). (B,D) High magnification of the region highlighted by dotted boxes in (A,C), respectively. hlx1 is expressed by tip and stalk ISV endothelial cells in std-MO injected embryos (arrows in B) whereas its expression is downregulated in ackr3b knockdown embryos (arrows in D). Scale bar: 100 μm.

ackr3b knockdown does not affect somite boundary development. Fluorescence analysis of 28 hpf Tg(kdrl:EGFP) zebrafish embryos injected with std-MO (A,C,E) or ackr3b-MO1 (B,D,F) after phalloidin staining to reveal somitic actin fibers (red in A,B,E,F). Note that somitic boundary formation occurs normally in ackr3b morphants also in the presence of impaired ISV patterning (white asterisks in D). Scale bar: 100 μm.

ackr3b knockdown causes alterations of blood circulation. (A–D)Tg(kdrl:EGFP;gata1:DsRed) embryos injected with std-MO (A,C) or ackr3b-MO1 (B,D) were photographed at 2 dpi under an epifluorescence microscope. Gata1⁺ elements were absent in Kdrl⁺ ISVs of ackr3b morphants (asterisks in B,D). Scale bar: 100 μm.

cxcr4a knockdown affects ISV patterning. Tg(kdrl:EGFP) embryos injected with std-MO (A,C,E,F) or cxcr4a-MO (B,D,G,H) were photographed at 26 hpf under an epifluorescence microscope. (C,D) Extended focus of z-stacks images representing high magnification of the trunk region. Asterisk in (D) indicates aberrant ISV branching in cxcr4a-MO-injected embryos. (E–H) Confocal microscopy analysis of 28 hpf Tg(kdrl:EGFP) zebrafish embryos injected with std-MO (E,F) or cxcr4a-MO (G,H). Control embryos show filopodia protrusions emerging from sprouting ISVs (E). At variance, filopodia protrusions are strongly reduced or absent in cxcr4a morphants (asterisk in H) despite ectopic ISV branching (G). High magnification of the regions highlighted by dotted boxes are shown in (E,G), respectively. (A–D) Scale bar: 100 μm. (E–G) Scale bar: 25 μm.

Setup of the μ-slide cell co-culture chemotaxis assay. (A) semi-quantitative PCR analysis of mock-CHO and ACKR3-CHO cells. (B) Microphotographs of EGFP-positive ACKR3-CHO cells. (C) Schematic representation of the μ-slide chamber. HUVECs (hereafter referred to as ECs) adherent to the central observation area are exposed to three different experimental conditions: no CXCL12 (–/–), addition of 50 ng/ml of CXCL12 to one (+/–) or both (+/+) lateral reservoirs. The horizontal arrow indicates the anticipated direction of HUVEC migration in the (+/–) experimental condition. (D) Quantification of forward migration index (FMI) and displacement of centre of mass (COM) parameters measured for HUVECs treated as illustrated in (C). (E) Schematic representation of the μ-slide chamber in the presence of mock-CHO cells. HUVECs were seeded in the central observation area whereas mock-CHO cells were seeded in both lateral reservoirs. After 4 h, cells were left untreated (–/–) or were incubated with 50 ng/ml CXCL12 added to one (+/–) or both (+/+) lateral reservoirs. (F) At the end of the incubation, FMI and COM parameters were calculated. (G) Schematic representation of the μ-slide chamber in the presence of ACKR3-CHO cells. HUVECs were seeded in the central observation area whereas ACKR3-CHO cells were seeded in both lateral reservoirs. After 4 h, cells were left untreated (–/–) or were incubated with 50 ng/ml CXCL12 added to one (+/–) or both (+/+) lateral reservoirs. At the end of the incubation, FMI and COM parameters were calculated (H). Data in panels (D,F,H) are the mean ± S.E.M of two independent experiments. ^*p < 0.05 or better, Student t-test.

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