miR-285–Yki/Mask double-negative feedback loop PNAS PLUS mediates blood–brain barrier integrity in Drosophila Dong Lia,b, Yanling Liua,b, Chunli Peic,d, Peng Zhangc,d, Linqing Pana,b, Jing Xiaoe, Songshu Mengb, Zengqiang Yuanc,d,1, and Xiaolin Bia,b,1 aDepartment of Biological Sciences, College of Basic Medical Sciences, Dalian Medical University, Dalian 116044, China; bInstitute of Cancer Stem Cell, Cancer Center, Dalian Medical University, Dalian 116044, China; cThe Brain Science Center, Beijing Institute of Basic Medical Sciences, Beijing 100850, China; dCenter of Alzheimer’s Disease, Beijing Institute for Brain Disorders, Beijing 100069, China; and eDepartment of Oral Basic Science, College of Stomatology, Dalian Medical University, Dalian 116044, China Edited by Norbert Perrimon, Harvard Medical School/HHMI, Boston, MA, and approved February 10, 2017 (received for review August 9, 2016) The Hippo signaling pathway is highly conserved from Drosophila to in size to maintain integrity of the BBB (7). Although an increased cell mammals and plays a central role in maintaining organ size and tissue size can be achieved through the accumulation of cell mass during the homeostasis. The blood–brain barrier (BBB) physiologically isolates growth of diploid cells, cell size is often correlated with the ploidy of the brain from circulating blood or the hemolymph system, and its DNA content and is increased via polyploidy during development, integrity is strictly maintained to perform sophisticated neuronal characteristics that are important for organogenesis, such as proper functions. Until now, the underlying mechanisms of subperineurial organ size, structure, and function (8–10). SPG cells have been shown glia (SPG) growth and BBB maintenance during development are to maintain the integrity of the BBB during development by increased not clear. Here, we report an miR-285–Yorkie (Yki)/Multiple Ankyrin ploidy with increased cell size (7). Despite its critical role in BBB repeats Single KH domain (Mask) double-negative feedback loop that formation and maintenance, the underlying mechanisms regulating regulates SPG growth and BBB integrity. Flies with a loss of miR-285 SPG cell growth and polyploidy are still poorly understood. have a defective BBB with increased SPG ploidy and disruptive sep- Previous studies have shown that Wnt/β-catenin and Sonic tate junctions. Mechanistically, miR-285 directly targets the Yki cofac- Hedgehog (SHH) signaling pathways are essential for BBB integrity tor Mask to suppress Yki activity and down-regulates the expression β (11). In Drosophila, Decapentaplegic (Dpp)/TGF- , Hedgehog BIOLOGY of its downstream target cyclin E, a key regulator of cell cycle. Dis- (Hh), and EGFR pathways promote proliferation and motility of DEVELOPMENTAL turbance of cyclin E expression in SPG causes abnormal endoreplica- glial cells (12, 13). The coactivation of EGFR and PI3K signaling tion, which leads to aberrant DNA ploidy and defective septate pathways in glia induces neoplasia (14). Recently, Yorkie (Yki), a junctions. Moreover, the expression of miR-285 is increased by knock- major effector of the Hippo pathway that regulates growth control, down of yki or mask and is decreased with yki overexpression, thus was reported to regulate proliferation of glial cells (15). Originally forming a double-negative feedback loop. This regulatory loop is identified in Drosophila and highly conserved from Drosophila to crucial for sustaining an appropriateYki/Maskactivityandcyclin E mammals, the Hippo signaling pathway plays a central role in level to maintain SPG ploidy and BBB integrity. Perturbation of this regulating organ size and tissue homeostasis. Central to this path- signaling loop, either by dysregulated miR-285 expression or Yki ac- way is a kinase cascade leading from Hippo to Yki (YAP and TAZ tivity, causes irregular SPG ploidy and BBB disruption. Furthermore, in mammals), ultimately inactivating Yki through phosphorylation miR-285 ectopic expression of promotes canonical Hippo pathway- and sequestering its subcellular localization from cytoplasm to nu- mediated apoptosis independent of the p53 or JNK pathway. Collec- cleus. In response to different intracellular or extracellular stimuli, tively, these results reveal an exquisite regulatory mechanism for BBB the Hippo pathway regulates cell proliferation, apoptosis, and miR-285– maintenance through an Yki/Mask regulatory circuit. stemness (16). Hippo | Mask | miR-285 | blood–brain barrier | subperineurial glia Significance o efficiently perform sophisticated neuronal functions, a well- The blood–brain barrier (BBB) is evolutionarily conserved from Tbalanced ion influx and efflux, as well as a steady supply of me- invertebrates to vertebrates to ensure a well-balanced ionic en- tabolites and nutrients, is required by the nervous system. To maintain vironment for proper neuronal functions. The Hippo pathway is a the homeostasis of ions and metabolites and prevent the transport of highly conserved signaling pathway essential for organ size con- neurotoxins and pathogens into the brain, the highly selective and trol and tissue homeostasis. Until now, whether Hippo pathway is permeable barrier called the blood–brain barrier (BBB) is evolu- required for BBB maintenance has been unknown. We show here tionarily conserved from invertebrates to vertebrates. The primitive that miR-285 is an upstream regulator of the Hippo pathway, BBB is formed at the embryonic stage and continues to mature after which can directly target Yorkie (Yki) cofactor Multiple Ankyrin birth. In higher order vertebrates, the BBB is formed primarily by the repeats Single KH domain (Mask). miR-285 and Yki/Mask form a brain vascular endothelium (1, 2); however, in Drosophila,theBBBis double-negative feedback loop to finely tune endoreplication of formed by two distinct classes of glial cells, perineurial glia (PG) and subperineurial glial (SPG) cells to keep proper cell size and maintain subperineurial glia (SPG). The apical PG cells form the first barrier to a functional BBB. Our findings propose an exquisite microRNA- prevent diffusion, and basal SPG cells form the extensive septate mediated regulatory circuit that regulates Hippo signaling activity junctions, a form of tight junctions, to prevent paracellular diffusion and tissue homeostasis during development. and are considered the structural basis of the BBB (3, 4). PG cells in Drosophila are not required to form the BBB during Author contributions: D.L., Z.Y., and X.B. designed research; D.L., Y.L., C.P., and L.P. performed research; P.Z., J.X., S.M., and Z.Y. contributed new reagents/analytic tools; early development, whereas SPG cells are essential for BBB mainte- D.L. and X.B. analyzed data; and D.L. and X.B. wrote the paper. nance during the early developmental stage and throughout develop- The authors declare no conflict of interest. ment to the adult stage (5). SPG cells form a flat, continuous layer and This article is a PNAS Direct Submission. tightly seal around the entire nervous system, and their proliferation is 1To whom correspondence may be addressed. Email: [email protected] or zyuan620@ restricted to embryogenesis (5, 6). During the larval stage, no addi- yahoo.com. tional SPG cells are generated, with the animals growing to a much This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. larger size; thus, SPG cells from the embryonic stage grow enormously 1073/pnas.1613233114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1613233114 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 As a transcription coactivator, Yki works with its major partner, miR-285 exhibit disrupted septate junctions and defective BBB. Sd, in flies to regulate the expression of global genes. Multiple miR-285 directly targets Yki cofactor Mask, and SPG cells in miR- Ankyrin repeats Single KH domain (MASK) protein is a newly 285KO flies have enhanced Yki activity and cyclin E expression, identified cofactor of Yki in Drosophila. By forming a complex with which leads to increased DNA ploidy, nuclear size in SPG cells, and Yki and Sd, Mask regulates cell proliferation and tissue growth brain hemisphere volume. These defects can be almost fully res- through positively modulating Yki activity and its downstream gene cued with the restricted expression of miR-285 or knockdown of expression (17, 18). YAP/TAZ can also regulate microRNA bio- mask or cyclin E expression. Furthermore, Yki/Mask forms a genesis in a cell density-dependent manner through modulation of double-negative feedback loop with miR-285 that is required for microRNA processing enzymes MicroprocessororDicercomplexes fine-tuning the DNA content in SPG during development. Thus, we (16, 19–21). In Drosophila, the microRNA bantam is a downstream provide direct evidence that the Hippo signaling pathway is re- target of Yki and is required for Yki-regulated cell growth (22, 23). quired for BBB integrity and identify an elaborate mechanism for bantam represses the Yki inhibitor SdBP/Tgi to establish a feedback BBB maintenance via the miR-285–Yki/Mask feedback signaling loop, which functionally mimics its mammalian homolog miR-130a loop, which is critical for the exquisite regulation of SPG polyploidy. targeting VGLL4, a YAP inhibitor (24). Although the core signaling cascade of the Hippo pathway has been extensively studied, whether Results Hippo pathway is functional at BBB maintenance is unknown, and miR-285 Is a Regulator of the Hippo Pathway by Targeting