The cAMP effector PKA mediates Moody GPCR signaling in Drosophila blood-brain barrier formation and maturation.

Xiaoling Li,Tina Schwabe,Liren Liu,Richard Fetter,Christophe Jung,Ulrike Gaul,Hermann Steller

doi:10.7554/elife.68275

Abstract

The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. Based on these findings, we propose that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical to maintain tissue size and brain homeostasis during organogenesis.

Highlights

The blood-brain barrier (BBB) is a complex physical barrier between the nervous system and the peripheral circulatory system that regulate CNS homeostasis to ensure proper neuronal function
To identify molecules that act downstream of Moody signaling in BBB formation, we examined genes known to be involved in Go protein-coupled receptor (GPCR) signaling, such as PkaC1, PI3K, PTEN, PLC, and Rap[1]
To rule out the possibility that the observed BBB defects are caused by glial cell fate or migration defects, we examined the presence and position of subperineural glia (SPG) using an antibody against the pan-glial, nuclear protein Reversed polarity (Repo) (Halter et al, 1995)

Summary

Introduction

The blood-brain barrier (BBB) is a complex physical barrier between the nervous system and the peripheral circulatory system that regulate CNS homeostasis to ensure proper neuronal function. We have previously identified a novel GPCR signaling pathway that is required for the proper organization of SJ belts between neighboring SPG at the embryonic stage, consisting of the receptor Moody, two hetero-trimeric G proteins (Gαiβγ, Gαoβγ), and the RGS protein Loco Both gain and loss of Moody signaling lead to non-synchronized growth of SPG cells, resulting in disorganized cell-contacts and shortened SJs and a leaky BBB (Schwabe et al, 2005; Schwabe et al, 2017). Our data reveal a previously unrecognized role of GPCR/PKA in maintaining enormous SPG cell growth and it’s sealing capability by regulating actomyosin contractility and the proper SJ organization in BBB formation and maturation, which touches the fundamental aspects of remodeling cytoskeletal network spatiotemporally - a common processes but with different mechanisms in morphogenesis

Results

Discussion

Experimental Procedures