Osteogenic suppressors such as Sclerostin not only regulate skeletal development and regeneration but also serve as anti-osteoporosis drug targets. However, very few druggable suppressors have been identified due to limited understanding of the molecular mechanisms governing osteogenesis. Here, we show that fibroblast activation protein (Fap), a serine protease inhibited by the bone growth factor Osteolectin, is an osteogenic suppressor. Genetic deletion of Fap significantly ameliorates limb trabecular bone loss during aging. Pharmacological inhibition of Fap significantly promotes bone formation and inhibits bone resorption in wild-type mice by differentially regulating canonical Wnt and nuclear factor κB (NF-κB) pathways. Pharmacological inhibition of Fap promotes osteoblast differentiation, inhibits osteoclast differentiation, and significantly attenuates osteoporosis in ovariectomized mice. Epistasis analyses in zebrafish show that Osteolectin functions as an endogenous inhibitor of Fap to promote vertebrae mineralization. Taken together, we identify Fap as an important osteogenic suppressor and a potential drug target to treat osteoporosis.

Phosphoinositides, a family of phosphorylated derivatives of phosphatidylinositol (PtdIns), are tightly regulated both temporally and spatially by PtdIns phosphatases and kinases. Mutations in inositol polyphosphate 5-phosphatase E (INPP5E) cause Joubert syndrome, a human disorder associated with numerous ciliopathic defects, including renal cyst formation, linking phosphoinositides to ciliopathies. However, the molecular mechanism by which INPP5E-mediated PtdIns signaling regulates ciliogenesis and cystogenesis is unclear. Here, we utilized an in vivo vertebrate model of renal cystogenesis to show that Inpp5e enzymatic activity at the apical membrane directs apical docking of basal bodies in renal epithelia. Knockdown or knockout of inpp5e led to ciliogenesis defects and cystic kidneys in zebrafish. Furthermore, knockdown of inpp5e in embryos led to defects in cell polarity, cortical organization of F-actin, and apical segregation of PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃. Knockdown of the ezrin gene, which encodes an ezrin/radixin/moesin (ERM) protein that crosslinks PtdIns(4,5)P₂ and F-actin, phenocopied inpp5e knockdowns. Notably, overexpression of the ezrin gene rescued inpp5e morphants. Finally, treatment with the PI 3-kinase inhibitor LY294002, which decreases PtdIns(3,4,5)P₃ levels, rescued the cellular, phenotypic, and renal functional defects in inpp5e-knockdown embryos. Together, our data indicate that Inpp5e functions as a key regulator of cell polarity in the renal epithelia, by inhibiting PtdIns(3,4,5)P₃ and subsequently stabilizing PtdIns(4,5)P₂ and recruiting Ezrin, F-actin, and basal bodies to the apical membrane, and suggest a possible novel approach for treating human ciliopathies.

P-TEFb activation by cAMP-PKA signaling promotes cystogenic gene transcription elongation and disease progression in ADPKD.

Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

Cyclin-dependent kinase 1 (CDK1) plays an essential role in cell cycle regulation. However, as mouse Cdk1 embryos die early, the role of CDK1 in regulating the cell cycle and embryo development remains unclear. Here, we showed that zebrafish cdk1-/- embryos exhibit severe microphthalmia accompanied by multiple defects in S phase entry, M phase progression, and cell differentiation but not in interkinetic nuclear migration. We identified Top2a as a potential downstream target and cyclin A2 and cyclin B1 as partners of Cdk1 in cell cycle regulation via an in silico analysis. While depletion of either cyclin A2 or Top2a led to the decreased S phase entry in zebrafish retinal cells, the depletion of cyclin B1 led to M phase arrest. Moreover, phosphorylation of Top2a at serine 1213 (S1213) was nearly abolished in both cdk1 and ccna2 mutants, but not in ccnb1 mutants. Furthermore, overexpression of TOP2AS1213D, the phosphomimetic form of human TOP2A, rescued S phase entry and alleviated the microphthalmia defects in both cdk1-/- and ccna2-/- embryos. Taken together, our data suggest that Cdk1 interacts with cyclin A2 to regulate S phase entry partially through Top2a phosphorylation and interacts with cyclin B1 to regulate M phase progression.

Polarity complexes, including the PAR (Partitioning‐defective), CRB (Crumbs) and SCRIB (Scribble) complexes, are required for the physiologic establishment, stabilization, and maintenance of a functional apical‐basolateral polarity. Inactivation of some of the polarity complexes results in cystic kidneys, and apical‐basolateral polarity defects are commonly observed in autosomal‐dominant polycystic kidney disease (ADPKD); however, little is known about the role that polarity complexes play in ADPKD. Here, we demonstrate that Scribble, a core protein of the SCRIB complex, is down‐regulated in ADPKD cell lines and the zebrafish model of this disease (pkd2 mor‐phants). Overexpression of Scribble could reduce cyst formation in pkd2 morphants, and loss of scrib led to a dilated pronephric duct in zebrafish. Furthermore, the Hippo signaling pathway was inactivated in scrib mutants and pkd2 morphants in which Yes‐associated protein (YAP), which is physiologically locatedinthe cytoplasm, was translocated to the nucleus. Of note, overexpression of cytoplasmic YAP, instead of nuclear YAP, could reduce cyst formation in pkd2 morphants. Consistently, knockout of yap resulted in cystic kidneys in zebrafish, which was rescued by the overexpression of cytoplasmic YAP, but not nuclear YAP. Finally, scrib and yap had a genetic interaction with pkd2 in cyst formation, and the overexpression of Scribble attenuated the down‐regulation of cytoplasmic YAP in ADPKD. Altogether, our data indicate that Scribble induces the phosphorylation of YAP and, consequently, influences cyst formation in ADPKD by mediating YAP nucleocytoplasmic shuttling.—Xu, D., Lv, J., He, L., Fu, L., Hu, R., Cao, Y., Mei, C. Scribble influences cyst formation in autosomal‐dominant polycystic kidney disease by regulating Hippo signaling pathway. FASEB J . 32, 4394–4407 (2018). www.fasebj.org