A Glial Interplay Regulates Junctional Architecture at the Blood–Brain Barrier of Drosophila

Neurons require a tightly controlled ion and metabolite homeostasis, which is maintained by an effective blood-brain barrier (BBB). Since the integrity of the BBB is essential for brain function, its maintenance during development represents a significant challenge. The Drosophila BBB is formed by two glial layers. Subperineurial glia (SPG) establish pleated septate junctions (pSJs) to generate the primary diffusion barrier. Abutting the SPG lies the mitotically active perineurial glia (PG). Only a small number of PG cells are specified during embryogenesis. The FGF8-like signal Pyramus presented by the SPG subsequently triggers proliferation of the PG, which ultimately covers the entire nervous system. To investigate the role of PG in the development and physiology of the BBB, we genetically ablated them. In these animals, glucose influx into SPG is unchanged following acute glucose perfusion. However, adult PG-ablated flies show increased sensitivity to glucose-only diet, indicating that while baseline carbohydrate transport can be maintained in the absence of most PG, the system fails under metabolic stress. SPG morphology and pSJ organization are largely preserved during larval stages upon PG ablation. In contrast, in PG-ablated pupae, moody expression is impaired in SPG and pSJs progressively split, producing ectopic junctional strands. Together, these results reveal the necessity of PG for the maturation and function of SPG, as well as the mutual interdependence between the two BBB glial layers.