Essential role for the d-Asb11 cul5 Box domain for proper notch signaling and neural cell fate decisions in vivo

Abstract

ECS (Elongin BC-Cul2/Cul5-SOCS-box protein) ubiquitin ligases recruit substrates to E2 ubiquitin-conjugating enzymes through a SOCS-box protein substrate receptor, an Elongin BC adaptor and a cullin (Cul2 or Cul5) scaffold which interacts with the RING protein. In vitro studies have shown that the conserved amino acid sequence of the cullin box in SOCS-box proteins is required for complex formation and function. However, the in vivo importance of cullin boxes has not been addressed. To explore the biological functions of the cullin box domain of ankyrin repeat and SOCS-box containing protein 11 (d-Asb11), a key mediator of canonical Delta-Notch signaling, we isolated a zebrafish mutant lacking the Cul5 box (Asb11(Cul)). We found that homozygous zebrafish mutants for this allele were defective in Notch signaling as indicated by the impaired expression of Notch target genes. Importantly, asb11(Cul) fish were not capable to degrade the Notch ligand DeltaA during embryogenesis, a process essential for the initiation of Notch signaling during neurogenesis. Accordingly, proper cell fate specification within the neurogenic regions of the zebrafish embryo was impaired. In addition, Asb11(Cul) mRNA was defective in the ability to transactivate a her4::gfp reporter DNA when injected in embryos. Thus, our study reporting the generation and the characterization of a metazoan organism mutant in the conserved cullin binding domain of the SOCS-box demonstrates a hitherto unrecognized importance of the SOCS-box domain for the function of this class of cullin-RING ubiquitin ligases and establishes that the d-Asb11 cullin box is required for both canonical Notch signaling and proper neurogenesis.

Figure 1. Schematic representation of Asb11 proteins.

(A), Asb11 functions as a substrate recognition module in a putative Elongin BC-Cullin-SOCS-box (ECS) type E3 ubiquitin ligase complex. (B), Sequence alignment of conserved Asb11 SOCS-box domain in different species. The cul5-box consensus sequence is indicated below the alignment. Identical amino acids are highlighted in red and similar ones in yellow. Dr: Danio rerio; Mm: Mus musculus; Hs: Homo sapiens. (C), (left) Illustration of the wild type and mutant d-asb11 gene products. Mutated protein is represented as Asb11^cul showing the predicted residual fragment and the position of the identified mutation. The different domains are indicated. (right) The T→A mutation changes a leucine into a stop codon.

Figure 2. Phenotypic assays on wild type and asb11^cul embryos.

(A), Morphological analysis of wild type and mutant embryos at 48 and 72hpf. (B), (left) Anterior view of wild type and mutant embryos at 10hpf after whole mount in situ hybridization, WISH, using probe against d-asb11. (right) Graph shows the quantification of the respective expressions using qPCR. (C), (left) Endogenous d-Asb11 in wild type (WT), heterozygous (asb11+/−) and mutant (asb11^cul) embryos at 12 hpf was detected by immunoblotting using anti-d-Asb11 antibody. (right) Graph quantifies 3 individual experiments, with 30 embryos/genotype/experiment.

Figure 3. Cullin box domain promotes induction of hes1 gene in vitro.

nTera-d1 cells were co-transfected with hes1-luciferase (hes1) or hes1-luciferase lacking the conserved CSL-binding site (hes1-RBPdel) and myc-tag (MT) as a control, or myc-tagged d-asb11 full length (MT-Asb11) or myc-tagged asb11^cul (MT-Asb11^cul) cDNA. Hes1-dependent Notch activity was analyzed by luciferase measurement.

Figure 4. asb11^cul presented altered expression of Delta-Notch pathway components.

Wild type (left panel) and mutant (middle panel) embryos at 12 hpf were analyzed for WISH using probes against her1, A; her4, B; her5, C; notch3, D; deltaD, E; and deltaA, F. (G), Higher magnification shows detailed analysis of deltaA expression. (left) Graphs quantify the mRNA expression levels.

Figure 5. her4::gfp transactivation and premature differentiation of neural cells in asb11^cul.

(A), the her4::gfp reporter was co-injected with myc-tag (MT) mRNA as a control, myc-tagged d-asb11 full length (MT-Asb11) or myc-tagged asb11^cul (MT-Asb11^cul) mRNA in zebrafish embryos. Injected embryos were treated with (+) (n = 25) or without (−) (n = 25) DAPT, from 1.5 hpf. At 14 hpf, embryos were analyzed for her4 transactivation based on the intensity of the GFP signal. Positive embryos were counted and percentages of embryos presenting weak (blue), medium (green) or strong (red) signal were given. (B), Wild type (left panel) and mutant (middle panel) embryos at 12 hpf were analyzed for WISH using probe against ngn1. (right) Graph quantifies expression of ngn1 using qPCR. (C) Wild type (left panel) and mutant (right panel) polster of embryos at 16 hpf were analyzed for WISH using probe against islet1.

Figure 6. Cullin box is essential for DeltaA degradation and for maintaining a cell proliferating state in vivo.

(A) Zebrafish embryos were injected with Myc-tagged deltaA (MT-DeltaA) and d-asb11 (Asb11) or asb11^cul (Asb11^cul) mRNA at one-cell stage. (lower panel) Lysates of 12 hpf embryos were analyzed by immunoblotting for the presence of DeltaA. (higher panel) Graph quantifies 2 individual experiments, each with 30 injected embryos/group. (B), Fluorescent whole-mount antibody labeling of wild type (WT) and asb11^cul embryos at 24 hpf for the mitotic marker anti-phosphohistone-3 (PH 3) antibody (green) and the neuronal marker Hu(C). Graph shows the number of positive cells per area (5 somites from beginning of yolk extension) of 5 embryos for each genotype.