N-methyl-N-nitrosourea (MNU) is a prevalent environmental carcinogen, which leads to tumors in various organs in animal models, while the mechanisms involved were still not fully understood. It is well known that anomalous angiogenesis is a key step in tumorigenesis and progression. In this study, we found that MNU induced abnormal angiogenesis which was accompanied by upregulation of rspo1, p53 and vegfaa in zebrafish embryos. Moreover, it revealed that MNU-induced ectopic sprouting of blood vessels was significantly reduced in rspo1knockdown but not p53-knockdown embryos, indicating that rspo1 was necessary for MNU-induced abnormal angiogenesis. Additionally, pharmaceutical activation or inhibition of Wnt/beta-catenin signaling pathway using (2'Z,3'E)- 6-bromoindirubin-3'-oxime or CCT036477 significantly increased or inhibited the pro-angiogenic effect of MNU on developing zebrafish embryos, which was confirmed by the effect of proliferation and migration in MNU-treated bEnd.3 cells. These data together indicated that rspo1/Wnt/beta-catenin/vegfaa axis is involved in the modulation of MNU-induced anomalous angiogenesis.

The blood-brain barrier (BBB) protects the central nervous system (CNS) from harmful elements, while it also restricts efficient drug delivery into the CNS. Previously, we generated a mutated fragment of Clostridium perfringens enterotoxin (cCPEYWSH ) which specifically binds to the endothelial tight junction protein claudin-5. Here, we explore the mechanisms regulating the dynamics of membranous claudin-5 and BBB permeability. Following cCPEYWSH binding to claudin-5, caveolin-1 mediates the redistribution of claudin-5 to the cytosol. This abnormal cytosolic aggregation triggers the autophagic degradation of claudin-5, leading to an increase in BBB permeability. Enhancement or inhibition of autophagy accelerates or inhibits the degradation of cytosolic claudin-5, respectively. Our findings may pave the way for improving BBB permeability for drug delivery.

Hypoxic injury to cerebrovascular endothelial cells (ECs) after stroke leads to blood-brain barrier (BBB) dysfunction, which is commonly associated with disruptions of endothelial tight junctions (TJs) and increased permeability. Therefore, maintaining the structural integrity and proper function of the BBB is essential for the homeostasis and physiological function of the central nervous system (CNS). Our previous study revealed that autophagy functions on protecting the BBB by regulating the dynamics of Claudin-5, the essential TJ protein, under short-term starvation or hypoxia conditions. Here, we show that in zebrafish and in vitro cells, loss of membranous Claudin-5 conversely determine the occurrence of hypoxia-induced autophagy in cerebrovascular ECs. Absence of endothelial Claudin-5 could partly attenuate endothelial cell apoptosis caused by short-term hypoxic injury. Mechanism studies revealed that under hypoxic conditions, the existence of membranous Claudin-5 affects the stimulation of hypoxia inducible factor 1 subunit alpha (HIF-1a) and the inducible nitric oxide synthase (iNOS), which are responsible for the translocation of and endocytosis of caveole-packaged Claudin-5 into cytosol. Meanwhile, loss of Claudin-5 affects the generation of reactive oxygen species (ROS) and the downstream expression of BCL2/adenovirus E1B 19kDa protein interacting protein 3 (Bnip3). These together suppress the endothelial autophagy under hypoxia. This finding provides a theoretical basis for clarifying the mechanism of hypoxia-induced BBB injury and its potential protection mechanisms.

The primary cilium, an important microtubule-based organelle, protrudes from nearly all the vertebrate cells. The motility of cilia is necessary for various developmental and physiological processes. Phosphoinositides (PIs) and its metabolite, PtdIns(4,5)P2, have been revealed to contribute to cilia assembly and disassembly. As an important kinase of the PI pathway and signaling, phosphatidylinositol 4-kinase β (PI4KB) is the one of the most extensively studied phosphatidylinositol 4-kinase isoform. However, its potential roles in organ development remain to be characterized. To investigate the developmental role of Pi4kb, especially its function on zebrafish ciliogenesis, we generated pi4kb deletion mutants using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 technique. The homozygous pi4kb mutants exhibit an absence of primary cilia in the inner ear, neuromasts, and pronephric ducts accompanied by severe edema in the eyes and other organs. Moreover, smaller otic vesicle, malformed semicircular canals, and the insensitivity on sound stimulation were characteristics of pi4kb mutants. At the protein level, both in vivo and in vitro analyses revealed that synthesis of Pi4p was greatly reduced owing to the loss of Pi4kb. In addition, the expression of the Pi4kb-binding partner of neuronal calcium sensor-1, as well as the phosphorylation of phosphatidylinositol-4-phosphate downstream effecter of Akt, was significantly inhibited in pi4kb mutants. Taken together, our work uncovers a novel role of Pi4kb in zebrafish inner ear development and the functional formation of hearing ability by determining hair cell ciliogenesis.