Abstract
Chemokine receptor CXCR4, its ligand stromal cell-derived factor-1 (CXCL12) and the decoy receptor atypical chemokine receptor 3 (ACKR3, also named CXCR7), are involved in the guidance of migrating cells in different anatomical districts. Here, we investigated the role of the ACKR3 zebrafish ortholog ackr3b in the vascularization process during embryonic development. Bioinformatics and functional analyses confirmed that ackr3b is a CXCL12-binding ortholog of human ACKR3. ackr3b is transcribed in the endoderm of zebrafish embryos during epiboly and is expressed in a wide range of tissues during somitogenesis, including central nervous system and somites. Between 18 somite and 26 h-post fertilization stages, the broad somitic expression of ackr3b becomes restricted to the basal part of the somites. After ackr3b knockdown, intersomitic vessels (ISVs) lose the correct direction of migration and are characterized by the presence of aberrant sprouts and ectopic filopodia protrusions, showing downregulation of the tip/stalk cell marker hlx1. In addition, ackr3b morphants show significant alterations of lateral dorsal aortae formation. In keeping with a role for ackr3b in endothelial cell guidance, CXCL12 gradient generated by ACKR3 expression in CHO cell transfectants guides human endothelial cell migration in an in vitro cell co-culture chemotaxis assay. Our results demonstrate that ackr3b plays a non-redundant role in the guidance of sprouting endothelial cells during vascular development in zebrafish. Moreover, ACKR3 scavenging activity generates guidance cues for the directional migration of CXCR4-expressing human endothelial cells in response to CXCL12.
Highlights
Chemokines and chemokine receptors, originally identified as mediators of the immune response, play non-redundant roles in various developmental processes [1]
Vasculogenesis occurs normally in ackr3b morphants as indicated by the presence of dorsal aorta (DA) and posterior cardinal vein (PCV). (F–I) Confocal microscopy analysis of 28 hpf Tg(kdrl:EGFP) zebrafish embryos injected with Standard MO (std-MO) (F,G) or ackr3b-MO1 (H,I)
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
Summary
Chemokines and chemokine receptors, originally identified as mediators of the immune response, play non-redundant roles in various developmental processes [1]. The chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1, known as CXCL12) are involved in the guidance of cell migration in several anatomical districts, including neuronal patterning [2,3,4]. CXCR7 Cues in Cell Migration networks, including gut, kidney and retinal vascularization in mice [5,6,7,8], as well as lateral dorsal aortae (LDA) patterning [9] and hindbrain capillary network formation [10] in zebrafish (Danio rerio). The two ackr genes identified in zebrafish, named ackr3a and ackr3b [2], modulate cell guidance cues during posterior lateral line formation [13,14,15,16], trigeminal sensory neuron migration [17] and primordial germ cell migration [18] during embryonic development
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