Mindbomb 2 is dispensable for embryonic development and Notch signalling in zebrafish

Abstract

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.

Keywords: Mib2; Neuronal development; Notch signalling; Ubiquitin ligase; Zebrafish.

Fig. 1.

Generation of mib2 mutants by TALEN. (A) Schematic representation of the genomic structure of the mib2 gene and mutations produced by TALEN. The TALEN target sequences are boxed. (B) Domain organization of Mib2 protein. All three mutations (cd1, cd2, and cd3) generate premature stop codons.

Fig. 2.

Expression of mib2 is reduced in mib2 mutant embryos. (A) Whole-mount in situ hybridization using the mib2 antisense probe in embryos at 48 hpf. Arrowheads indicate mib2 expression in the ear. Head region, side views of embryos at 48 hpf with anterior to the left. (B) Relative expression level of mib2 mRNA measured by q-PCR in the 48 hpf-embryos. Error bars represent the mean±s.d. of three independent experiments.

Fig. 3.

Mib2 deficiency does not significantly affect early neurogenesis and Notch signalling. Whole-mount in situ hybridization using elavl3 (A) and her4.1 (B) antisense probes at the 3 somite stage, showing no difference in neurogenesis phenotypes between wild-type and mib2^cd3 embryos or between the mib1 mutants and mib1/mib2 double mutants. All panels show top views of embryos with the anterior to the left.

Fig. 4.

Maternal Mib2 does not compensate for the zygotic loss of Mib2. Whole-mount in situ hybridization using her4.1 (A) and xirp2a (B) antisense probe at 24 hpf. Maternal deletion of mib2 does not affect expression of her4.1 or xipr2a. Whole embryo (A) and trunk region (B) are shown. Arrows show expression of her4 in the trunk neural tube. Side views of embryos with anterior to the left.

Fig. 5.

Mib2 neither regulates melanophore development nor antagonizes Mib1^ta52b protein. Whole embryos (A) or enlarged views (B) of tail region in A. mib2 deletion did not enhance pigmentation loss in mib1^tfi91, nor did it recover pigmentation in mib1^ta52b mutants. All panels show side views of embryos with anterior to the left.

Fig. 6.

Muscle structure is normal in mib2 mutant embryos. (A) Whole-mount in situ hybridization using the myoD in trunk region. (B) Slow muscle fibre myosin in trunk muscles as revealed by F59 antibody. mib2 mutant embryos did not show any changes in the pattern or level of their staining. All panels show side views of embryos with anterior to the left at 24 hpf.

Fig. 7.

Mib2 deficiency does not affect survival of 4 dpf embryos exposed NH₄Ac. Embryos were monitored for their heartbeats every 30 min. NaAc, sodium acetate-treated groups. NH₄Ac, ammonium acetate-treated groups. The survival of mib2^cd3 embryos was comparable to that of wild type control embryos by NH₄Ac treatment. NaAC wild type, n=14; NH₄Ac wild type, n=15. NaAC mib2^cd3, n=7; NH₄Ac mib2^cd3, n=8.