Objective To determine the effect of knocking down zebrafish faf1 gene by CRISPR/Cas9 editing technique.Methods gRNA was designed and prepared for the faf1 gene of zebrafish,and gRNA was mixed with Cas9 mRNA by microinjection into zebrafish single cell embryos.The mutant F0 generation zebrafish was screened out by enzyme digestion and gene sequencing.The mutant F0 was genetically outcrossed with the wild-type zebrafish to get the F1 heterozygous zebrafish,and the genotype of zebrafish was detected by microscopic observation.Results The faf1 gRNA and Cas9 mRNA were successfully prepared.The gRNA (gRNA6) located in the exon 6 of faf1 could shift the faf1 gene into frameshift mutations.The mutation type MU1 was screened out and the somatic cytochrome deposition delay was observed in this heterozygous zebrafish.At 4 d post fertilization (dpf),there were sarcomeric dysplasia and head shrinkage,increased hyoid angle and other craniofacial cartilage deformities.And the zebrafish died at 8 ～9 dpf.Conclusion CRISPR/Cas9 knocking out thefaf1 gene produces a new phenotype for zebrafish,with delayed pigment deposition and nodule-like change in tail muscle section.

Amyloid beta (Aβ) precursor protein (APP) is a key protein in the pathogenesis of Alzheimer's disease (AD). Both APP and its paralogue APLP1 (amyloid beta precursor-like protein 1) have multiple functions in cell adhesion and proliferation. Previously it was thought that autophagy is a novel beta-amyloid peptide (Aβ)-generating pathway activated in AD. However, the protein proteolysis of APLP1 is still largely unknown. The present study shows that APLP1 is rapidly degraded in neuronal cells in response to stresses, such as proteasome inhibition. Activation of the endoplasmic reticulum (ER) stress by proteasome inhibitors induces autophagy, causing reduction of mature APLP1/APP. Blocking autophagy or JNK stress kinase rescues the protein expression for both APP and APLP1. Therefore, our results suggest that APP/APLP1 is degraded through autophagy and the APLP1 proteolysis is mainly mediated by autophagy-lysosome pathway.
Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Autophagy ; Cell Line ; Endoplasmic Reticulum ; metabolism ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Leupeptins ; pharmacology ; Mice ; Neurons ; cytology ; metabolism ; Proteasome Endopeptidase Complex ; metabolism ; Proteasome Inhibitors ; Protein Stability ; Rats

We investigate the role of β-catenin signaling in the response of macrophage to lipopolysaccharide (LPS) using RAW264.7 cells. LPS rapidly stimulated cytosolic β-catenin accumulation. β-catenin-mediated transcription was showed to be required for LPS induced gene expression and cell migration. Mechanically, ERK activation-primed GSK3β inactivation by Akt was demonstrated to mediate the LPS induced β-catenin accumulation. Overall, our findings suggest that suppression of GSK3β by ERK stimulates β-catenin signaling therefore contributes to LPS induced cell migration in macrophage activation.
Animals ; Cell Line ; Cell Movement ; drug effects ; Enzyme Activation ; drug effects ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Gene Expression Regulation, Enzymologic ; drug effects ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Lipopolysaccharides ; pharmacology ; Macrophages ; cytology ; drug effects ; enzymology ; metabolism ; Matrix Metalloproteinase 9 ; genetics ; Mice ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction ; drug effects ; Transcription, Genetic ; drug effects ; Wnt Proteins ; metabolism ; beta Catenin ; metabolism

The Wnt/β-catenin and bone morphogenetic proteins (BMPs) pathways play important roles in controlling osteogenesis. Using a cell-based kinase inhibitor screening assay, we identified the compound bisindoylmaleimide I (BIM) as a potent agonist of the cytosolic β-catenin accumulation in preosteoblast cells. Through suppressing glycogen synthase kinase 3β enzyme activities, BIM upregulated β-catenin responsive transcription and extended duration of BMP initiated signal. Functional analysis revealed that BIM promoted osteoblast differentiation and bone formation. The treatment of human mesenchymal stem cells with BIM promoted osteoblastogenesis. Our findings provide a new strategy to regulate mesenchymal stem cell differentiation by integration of the cellular signaling pathways.
Animals ; Bone Morphogenetic Proteins ; metabolism ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; Indoles ; chemistry ; pharmacology ; Maleimides ; chemistry ; pharmacology ; Mesenchymal Stem Cells ; cytology ; metabolism ; Mice ; Osteoblasts ; cytology ; drug effects ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Signal Transduction ; drug effects ; Wnt Proteins ; metabolism ; beta Catenin ; antagonists & inhibitors ; genetics ; metabolism

Microtubules play important roles in mitotic spindle assembly and chromosome segregation to maintain normal cell cycle progression. A number of microtubule-associated proteins have been identified in epithelial and neural cell cultures; however, their physiological significance is not well characterized due to the lack of appropriate in vivo animal models. Nucleolar spindle-associated protein (NuSAP) is a microtubule-binding protein and is reported to be involved in mitosis by cell culture studies. In this report, we identified the zebrafish homologue of human NuSAP and investigated its expression profile and functions. Using in situ hybridization, we demonstrated that transcripts of zebrafish nusap1 are specifically expressed in the retina, forebrain, hindbrain and neural crest. When the in vivo expression of nusap1 was knocked down through antisense oligonucleotide morpholino technology, the morphants of nusap1 showed impaired morphogenesis in the trunk and yolk extension, implying the involvement of Nusap1 in cell migration. Mechanistic studies revealed that nusap1 morphants have an altered expression pattern of neural crest markers crestin and sox9b, but normal expression of blood vessel and notochord markers gata1 and shh. In addition, nusap1 mRNA injection caused serious apoptosis in retina and hindbrain tissue, and these phenotypes can be rescued by co-injection of morpholino against nusap1. These observations not only suggest a role for Nusap1 in connecting apoptosis with cell migration, but also provide strong evidences that Nusap1 is potentially involved in morphogenesis in vertebrates.
Amino Acid Sequence ; Animals ; Animals, Genetically Modified ; Apoptosis ; genetics ; physiology ; Base Sequence ; Cell Movement ; genetics ; physiology ; Cloning, Molecular ; DNA Primers ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Humans ; In Situ Hybridization ; Microtubule-Associated Proteins ; genetics ; physiology ; Molecular Sequence Data ; Neural Crest ; cytology ; embryology ; physiology ; Phylogeny ; Sequence Homology, Amino Acid ; Zebrafish ; embryology ; genetics ; Zebrafish Proteins ; genetics ; physiology