Apical PtdIns(4,5)P2 is required for ciliogenesis and suppression of polycystic kidney disease.

Cilia are hair-like structures that function like antennae to detect chemical and mechanical signals in the environment. Recently, phosphoinositides were shown to play an important role in cilia assembly and disassembly. However, the precise molecular and cellular mechanisms underlying this process remain unknown. Here, we report that suppression of apical phosphatidylinositol 4,5- bisphosphate [PtdIns(4,5)P2], by overexpressing apically targeted PtdIns(4,5)P2 phosphatase or by knocking down type I phosphatidylinositol 4-phosphate 5-kinase (Pip5k1), leads to ciliogenesis defects and polycystic kidney disease (PKD) in zebrafish embryos that phenocopied inpp5e mutant, a Joubert syndrome model. We further demonstrate that decreased expression of apical PtdIns(4,5)P2 disrupted apical ezrin recruitment, F-actin organization, and basal body docking. Moreover, the ciliogenesis and polycystic kidney defects in PtdIns(4,5)P2-depleted embryos can be rescued by overexpression of ezrin. Finally, Pip5k1a overexpression rescued the ciliogenesis defects and PKD phenotypes in Inpp5e-depleted embryos. Taken together, our results reveal that apical PtdIns(4,5)P2 is essential for ciliogenesis and the prevention of PKD and suggest a novel possibility for treating PKD and other human ciliopathies.-Xu, W., Jin, M., Huang, W., Wang, H., Hu, R., Li, J., Cao, Y. Apical PtdIns(4,5)P2 is required for ciliogenesis and suppression of polycystic kidney disease.

Mutagenesis of putative ciliary genes with the CRISPR/Cas9 system in zebrafish identifies genes required for retinal development.

Cilia are conserved microtubule-based organelles that function as mechanical and chemical sensors in various cell types. By bioinformatic, genomic, and proteomic studies, more than 2000 proteins have been identified as cilium-associated proteins or putative ciliary proteins; these proteins are referred to as the ciliary proteome or the ciliome. However, little is known about the function of these numerous putative ciliary proteins in cilia. To identify the possible new functional proteins or pathways in cilia, we carried out a small-scale genetic screen targeting 54 putative ciliary genes by using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system. We successfully constructed 54 zebrafish mutants, and 8 of them displayed microphthalmias. Three of these 8 genes encode proteins for protein transport, suggesting the important roles of protein transport in retinal development. In situ hybridization revealed that all these genes are expressed in zebrafish eyes. Furthermore, polo-like kinase 1 was required for ciliogenesis in neural tube. We uncovered the potential function of the ciliary genes for the retinal development of zebrafish.-Hu, R., Huang, W., Liu, J., Jin, M., Wu, Y., Li, J., Wang, J., Yu, Z., Wang, H., Cao, Y. Mutagenesis of putative ciliary genes with the CRISPR/Cas9 system in zebrafish identifies genes required for retinal development.

Single-Cell RNA-Sequencing Atlas Reveals the Tumor Microenvironment of Metastatic High-Grade Serous Ovarian Carcinoma.

Ovarian cancer is the most common and lethal gynecological tumor in women worldwide. High-grade serous ovarian carcinoma (HGSOC) is one of the histological subtypes of epithelial ovarian cancer, accounting for 70%. It often occurs at later stages associated with a more fatal prognosis than endometrioid carcinomas (EC), another subtype of epithelial ovarian cancer. However, the molecular mechanism and biology underlying the metastatic HGSOC (HG_M) immunophenotype remain poorly elusive. Here, we performed single-cell RNA sequencing analyses of primary HGSOC (HG_P) samples, metastatic HGSOC (HG_M) samples, and endometrioid carcinomas (EC) samples. We found that ERBB2 and HOXB-AS3 genes were more amplified in metastasis tumors than in primary tumors. Notably, high-grade serous ovarian cancer metastases are accompanied by dysregulation of multiple pathways. Malignant cells with features of epithelial-mesenchymal transition (EMT) affiliated with poor overall survival were identified. In addition, cancer-associated fibroblasts with EMT-program were enriched in HG_M, participating in angiogenesis and immune regulation, such as IL6/STAT3 pathway activity. Compared with ECs, HGSOCs exhibited higher T cell infiltration. PRDM1 regulators may be involved in T cell exhaustion in ovarian cancer. The CX3CR1_macro subpopulation may play a role in promoting tumor progression in ovarian cancer with high expression of BAG3, IL1B, and VEGFA. The new targets we discovered in this study will be useful in the future, providing guidance on the treatment of ovarian cancer.

Cyclin A2/cyclin-dependent kinase 1-dependent phosphorylation of Top2a is required for S phase entry during retinal development in zebrafish.

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.

Anti-obesity potential of rare sugar d-psicose by regulating lipid metabolism in rats.

d-Psicose is a new-generation sugar substitute with a low calorie count and can still offer the desirable sweetness. The objective of this study was to investigate the antiobesity potential of d-psicose and the possible mechanism using Wistar rats as the animal model. The animals were divided into five groups and supplemented with diets containing 5% of different carbohydrates, such as glucose, fructose, cellulose, d-psicose, and a control diet, for 4 weeks. After sacrifice, blood lipid profile, tissue morphology, and related genes participating in lipid metabolism were analyzed. The results indicated that the supplementation by d-psicose leads to minimum fat accumulation in rats when compared with the other carbohydrates. The blood lipid profile and antioxidative activity of the rat were also improved. d-Psicose can regulate lipid metabolism by increasing the lipid-metabolism-related enzymes such as SDH in serum and liver and HL in the liver. d-Psicose can prevent fat accumulation by suppressing the expression of lipogenesis-related gene ACCα and hepatic fatty acid uptake gene (FAS and SREBP-1c), while stimulating the expression for fatty-acid-oxidation-related gene including AMPK2α, HSL, and PPARα. In conclusion, d-psicose can be considered to be a healthy alternative to traditional sweeteners.

Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

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.