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 mibtfi91-null phenotypes nor did it alleviate antimorphic mibta52b 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.

Gene knockdowns using oligonucleotide-based approaches are useful for studying gene function in both in vitro cell culture systems and in vivo animal models. We evaluated the efficacy of 2',4'-bridged nucleic acids (BNA)-modified antisense oligonucleotides (AONs) for gene knockdown in zebrafish. We used the tcf7l1a gene as a model for testing the knockdown efficacy of 2',4'-BNA AONs and examined how the target sites/affinity and RNase H induction activity of 2',4'-BNA AONs affect knockdown efficacy. We found that tcf7l1a gene function was knocked down by 2',4'-BNA AONs that target the start codon and induce RNase H activity. Although nonspecific p53-mediated developmental defects were observed at higher doses, the effective dose of the 2',4'-BNA AONs for tcf7l1a is much lower than that of morpholino oligonucleotides. Our data thus show a potential application for 2',4'-BNA AONs in the downregulation of specific genes in zebrafish.