In this work, we aim to construct a new behavior analysis method by using machine learning. We used two cameras to capture three-dimensional (3D) tracking data of zebrafish, which were analyzed using fuzzy adaptive resonance theory (FuzzyART), a type of machine learning algorithm, to identify specific behavioral features. The method was tested based on an experiment in which electric shocks were delivered to zebrafish and zebrafish swimming was tracked in 3D simultaneously to find electric shock-associated behaviors. By processing the obtained data with FuzzyART, we discovered that distinguishing behaviors were statistically linked to the electric shock based on the machine learning algorithm. Moreover, our system could accept user-supplied data for detection and quantitative analysis of the behavior features, such as the behavior features defined by the 3D tracking analysis above. This system could be applied to discover new distinct behavior features in mutant zebrafish and used for drug administration screening and cognitive ability tests of zebrafish in the future.

Persistent directional cell migration is involved in animal development and diseases. The small GTPase Rac1 is involved in F-actin and focal adhesion dynamics. Local Rac1 activity is required for persistent directional migration, whereas global, hyperactivated Rac1 enhances random cell migration. Therefore, precise control of Rac1 activity is important for proper directional cell migration. However, the molecular mechanism underlying the regulation of Rac1 activity in persistent directional cell migration is not fully understood. Here, we show that the ubiquitin ligase mind bomb 1 (Mib1) is involved in persistent directional cell migration. We found that knockdown of MIB1 led to an increase in random cell migration in HeLa cells in a wound-closure assay. Furthermore, we explored novel Mib1 substrates for cell migration and found that Mib1 ubiquitinates Ctnnd1. Mib1-mediated ubiquitination of Ctnnd1 K547 attenuated Rac1 activation in cultured cells. In addition, we found that posterior lateral line primordium cells in the zebrafish mib1^ta52b mutant showed increased random migration and loss of directional F-actin–based protrusion formation. Knockdown of Ctnnd1 partially rescued posterior lateral line primordium cell migration defects in the mib1^ta52b mutant. Taken together, our data suggest that Mib1 plays an important role in cell migration and that persistent directional cell migration is regulated, at least in part, by the Mib1–Ctnnd1–Rac1 pathway. Significance Cell migration is involved in various biological processes, including animal development and cancer metastasis. Cells show either of two different types of migratory behavior: random or directional. We show that defects in mind bomb 1 (Mib1) ubiquitin ligase, an enzyme that adds ubiquitin to substrates, leads to an increase in random cell migration. Our study revealed that Mib1 ubiquitinates Ctnnd1, a positive regulator of the small GTPase Rac1, in cultured cells. We further found that Mib1-dependent Ctnnd1 ubiquitination decreases Ctnnd1-mediated activation of Rac1, which increases random cell migration, in cultured cells. Therefore, this study shows that Mib1 contributes to persistent directional cell migration, at least in part, by regulating the Ctnnd1–Rac1 pathway.

Glucocorticoid-induced osteoporosis (GIOP) is a major cause of secondary osteoporosis, and the pathogenic mechanisms of GIOP remain to be elucidated. Here, we show a rapid dexamethasone-induced osteoporosis animal model using zebrafish scales. Intraperitoneal injection of dexamethasone over a 5-day period suppressed the regeneration of scales. Furthermore, the circularity of the newly formed regenerated scales was also slightly reduced compared to that of the control group on day 5. The changes in bone-related enzymes, such as cathepsin K, tartrate-resistant acid phosphatase (TRAP) for bone resorption, and alkaline phosphatase (ALP) for bone formation, provide insight into the progression of bone diseases; therefore, we further developed a method to measure the activities of cathepsin K, TRAP, and ALP using zebrafish scales. We found that a lysis buffer with detergent at neutral pH under sonication efficiently helped extract these three enzymes with high activity levels. Interestingly, treatment with a dexamethasone injection produced considerably higher levels of cathepsin K activity and a lower Ca/P ratio than those in the control group, suggesting that dexamethasone increased osteoclast activity, with no significant changes in the activities of TRAP and ALP. Our GIOP model and enzyme assay method could help to design better treatments for GIOP.

The Mindbomb E3 ubiquitin protein ligase (Mib) family of proteins, Mib1 and Mib2, are RING finger ubiquitin ligases that share specific substrates. Mib1 is known to play essential roles in Notch signalling by ubiquitinating Notch ligands in vivo. Conversely, the functions of Mib2 in vivo are not fully understood, although Mib2 ubiquitinates multiple substrates, including Notch ligands, in vitro. To determine the Notch-dependent and Notch-independent functions of Mib2 in vivo, we generated mutant alleles of zebrafish mib2 using transcription activator-like effector nucleases (TALENs). We found that mib2 homozygous mutants were viable and fertile. Notch-mediated functions, such as early neurogenesis, somitogenesis, and pigment cell development, were not affected in mib2 mutant embryos. The lack of Notch-deficient phenotypes in mib2 mutants was not due to compensation by a mib2 maternal gene product because mib2 maternal-zygotic mutants also did not exhibit a distinct phenotype. We also showed that Mib2 does not redundantly act with Mib1 because the genetic ablation of mib2 neither enhanced mib^tfi91-null phenotypes nor did it alleviate antimorphic mib^ta52b phenotypes. Furthermore, the postulated Notch-independent roles of Mib2 in maintaining muscular integrity and N-methyl-D-aspartate receptor (NMDAR) activity were not evident: mib2 mutants did not show phenotypes different from that of the control embryos. These observations suggest that Mib2 is dispensable for embryonic development and does not have redundant functions with Mib1 in Notch signalling at least during early development stages in zebrafish.