Dynamic changes of extracellular vesicles during zebrafish organogenesis.

Extracellular Vesicles (EVs) play a crucial role in cell differentiation. Despite its role as a well-established vertebrate model, little is known about EVs during zebrafish embryogenesis. This study investigates EVs during zebrafish embryogenesis, analysing size- and concentration-changes over time.Wild-type AB strain zebrafish larvae (zfl) were collected at 24, 48, 72, and 96 h post fertilization (hpf) and homogenized. EVs were isolated and characterized using flow cytometry, negative staining transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western Blot.Flow cytometry and TEM showed a high purity of the samples. Small EVs (sEVs) and large EVs (lEVs) were differentiated using NTA, showing that zfl use different types of EVs during embryogenesis. It was observed that the total EV number increased significantly over the first 72 hpf, but not proportionally to zfl growth in length. Additionally, sEV size also increased significantly, with a maximum diameter at 72 hpf. Since most organs are formed during the first 72 hpf and from then on mainly maturation and growth occur, the elevated number and larger size before 72 hpf suggests an important role of EVs during zebrafish organogenesis. Since EVs serve as cargo delivery platforms, the increase in size of sEVs may reflect the need for a higher transport capacity.