Zebrafish glial-vascular interactions progressively expand over the course of brain development

Glial-vascular interactions are critical for the formation and maintenance of brain blood vessels and the blood-brain barrier (BBB) in mammals, but their role in zebrafish is not well understood. Our previous work has detailed the timeline of BBB functional maturation in zebrafish, revealing a conserved mechanism of BBB induction through the suppression of endothelial transcytosis. Yet, as opposed to extensive research on glial-vascular interactions in rodents, such interactions remain largely overlooked in the zebrafish model system. Here, we focus on glial-vascular development in the zebrafish brain, leveraging three glial gene promoters: gfap (glial fibrillary acidic protein), glast (an astrocyte-specific glutamate transporter), and glastini (a new, shortened, equally effective version of the Glast promoter). Using these glial promoters, sparse labeling revealed fewer glial-vascular interactions during early larval stages, with both glial coverage and contact area increasing as the zebrafish brain matured. We then generated stable transgenic lines for both the Glast and Glastini promoters and observed similar increases in glial coverage during larval development, starting at ∼30% coverage at 3 days post-fertilization (dpf) and peaking at ∼60% at 10 dpf. Ultrastructural assessment of glial-vascular interactions using electron microscopy (EM) confirmed a progressive increase in glial coverage over larval development, with maximal coverage reaching ∼70% in adult zebrafish, significantly lower than the nearly 100% coverage observed in mammals. Finally, immunogold-EM labeling confirmed that cells identified as glia in aforementioned morphological analyses were indeed Glast-positive. Taken together, our results identify the temporal profile of glial-vascular maturation in the zebrafish brain.