<i>miR-285-</i>Yki/Mask double-negative feedback loop mediates blood-brain barrier integrity in <i>Drosophila</i>.

Abstract

The Hippo signaling pathway is highly conserved from <i>Drosophila</i> to mammals and plays a central role in maintaining organ size and tissue homeostasis. The blood-brain barrier (BBB) physiologically isolates the brain from circulating blood or the hemolymph system, and its integrity is strictly maintained to perform sophisticated neuronal functions. Until now, the underlying mechanisms of subperineurial glia (SPG) growth and BBB maintenance during development are not clear. Here, we report an <i>miR-285</i>-Yorkie (Yki)/Multiple Ankyrin repeats Single KH domain (Mask) double-negative feedback loop that regulates SPG growth and BBB integrity. Flies with a loss of <i>miR-285</i> have a defective BBB with increased SPG ploidy and disruptive septate junctions. Mechanistically, <i>miR-285</i> directly targets the Yki cofactor Mask to suppress Yki activity and down-regulates the expression of its downstream target <i>cyclin E</i>, a key regulator of cell cycle. Disturbance of <i>cyclin E</i> expression in SPG causes abnormal endoreplication, which leads to aberrant DNA ploidy and defective septate junctions. Moreover, the expression of <i>miR-285</i> is increased by knockdown of <i>yki</i> or <i>mask</i> and is decreased with <i>yki</i> overexpression, thus forming a double-negative feedback loop. This regulatory loop is crucial for sustaining an appropriate Yki/Mask activity and <i>cyclin E</i> level to maintain SPG ploidy and BBB integrity. Perturbation of this signaling loop, either by dysregulated <i>miR-285</i> expression or Yki activity, causes irregular SPG ploidy and BBB disruption. Furthermore, ectopic expression of <i>miR-285</i> promotes canonical Hippo pathway-mediated apoptosis independent of the p53 or JNK pathway. Collectively, these results reveal an exquisite regulatory mechanism for BBB maintenance through an <i>miR-285</i>-Yki/Mask regulatory circuit.