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.
BMP signaling is critical for many biological processes. Therefore, small molecules that modulate BMP signaling are useful for elucidating the function of BMP signaling and treating BMP signaling-related diseases. Here, we performed a phenotypic screening in zebrafish to examine the in vivo effects of N-substituted-2-amino-benzoic acid analogs NPL1010 and NPL3008 and found that they affect BMP signaling-dependent dorsal–ventral (D–V) patterning and bone formation in zebrafish embryos. Furthermore, NPL1010 and NPL3008 suppressed BMP signaling upstream of BMP receptors. BMP1 cleaves Chordin, an antagonist of BMP, and negatively regulates BMP signaling. Docking simulations demonstrated that NPL1010 and NPL3008 bind BMP1. We found that NPL1010 and NPL3008 partially rescued the disruptions in the D–V phenotype caused by bmp1 overexpression and selectively inhibited BMP1-dependent Chordin cleavage. Therefore, NPL1010 and NPL3008 are potentially valuable inhibitors of BMP signaling that act through selective inhibition of Chordin cleavage.
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