HIROYUKI NAKAJIMA

The connection between the heart and great vessels established during embryogenesis is essential for circulation. However, how great veins adhere to the endocardium lining the inside lumen of the beating heart remains unknown. Here, using zebrafish, we demonstrate that the endocardium and great veins are sealed in a zipper-closing manner outside the beating heart. The gradual elongation of the endocardium, driven by convergent extension, organized this adhesion by pulling venous endothelial cells (ECs) along the anterior-posterior axis. Time-specific manipulation of the heart rate revealed that this endocardial elongation proceeds against heartbeat-driven force. From time-lapse imaging of adherens junctions, which would counterbalance mechanical forces, we found a specific contribution of cadherin-6 instead of cadherin-5 in sensing endocardium-specific mechanical force. This specificity was confirmed by the depletion of cadherin-6 that caused endocardium deformation. Altogether, we propose that cadherin-6-mediated EC-zippering updates the understanding of cadherin usage in dynamic morphogenesis.

Scavenger endothelial cells (SECs) lining the liver sinusoids play a critical role in the rapid blood clearance of nanoparticle (NP)-based drug-delivery systems. However, how these cells recognize synthetic materials is largely unknown, which hampers the establishment of NP design criteria for prolonging their blood circulation time upon systemic administration. This study investigates how the surface-grafted chain conformation on the NPs affects their uptake by SECs. Polystyrene NPs are grafted with polyethylene glycol (PEG) polymers having different molecular weights (MWs = 2, 5, 10, and 20 kDa). The conformation of surface PEG chains depends on their MWs, with PEG chains being in the mushroom regime for the lowest MW and transforming into a brush configuration with an increase in the MW. The surface PEG chains with a brush conformation inhibit cellular uptake of the NPs by a SEC model cell line SK-Hep-1 significantly, while NPs with a mushroom conformation are readily taken up. Furthermore, quantitative pharmacokinetic analysis in zebrafish larvae by combining a single-particle tracking technique and computer-aided image analysis reveals that NPs with longer PEG chains, especially for 20 kDa, show significantly prolonged blood circulation time due to avoiding clearance by SECs.

During angiogenesis, sprouting endothelial cells (ECs) migrate and eventually connect to target vessels to form new vessel branches. However, it remains unclear how these sprouting vessels migrate toward the target vessels in three-dimensional space. We performed in vivo imaging of the cerebral capillary network formation in zebrafish to investigate how sprouting tip cells migrate toward their targets. Of note, we found that tip cells reach the target vessels through two phases: a non-directional phase and a directional phase. In the non-directional phase, sprouting tip cells dynamically extend and retract their protrusions at the leading front and have less directionality in their movement. In contrast, once tip cells enter the directional phase, they migrate directly toward the anastomotic targets. Chemokine receptor Cxcr4a and its ligand Cxcl12b are important for the phase transition to the directional phase. In cxcr4a mutants, sprouting tip cells lose their directionality and tend to connect to nearby sprouting ECs, resulting in altered capillary network patterning. Furthermore, in wild-type (WT) larvae, local Ca2+ oscillations were detected in protrusions of tip cells, specifically in the non-directional phase, but almost disappeared in the directional phase as a result of the Cxcr4-dependent phase transition. Thus, this study provides evidence of a chemokine-induced phase transition in migrating tip cells, which is important for proper vascular network formation in the zebrafish brain.Key words: angiogenesis, directional migration, live imaging, chemokine, Ca2+ dynamics, zebrafish.