Phenotypic assays on wild type and <i>asb11^cul</i> embryos.

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

Cullin box domain promotes induction of <i>hes1</i> gene <i>in vitro</i>.

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

<i>her4::gfp</i> transactivation and premature differentiation of neural cells in <i>asb11^cul</i>.

<p>(<b>A</b>), the <i>her4::gfp</i> reporter was co-injected with <i>myc-tag</i> (MT) mRNA as a control, myc-tagged <i>d-asb11</i> full length (MT-Asb11) or myc-tagged <i>asb11^cul</i> (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 <i>her4</i> 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>B</b>), Wild type (<i>left panel</i>) and mutant (<i>middle panel</i>) embryos at 12 hpf were analyzed for WISH using probe against <i>ngn1</i>. (<i>right</i>) Graph quantifies expression of <i>ngn1</i> using qPCR. (<b>C</b>) Wild type (<i>left panel</i>) and mutant (<i>right panel</i>) polster of embryos at 16 hpf were analyzed for WISH using probe against <i>islet1</i>.</p

Cullin box is essential for DeltaA degradation and for maintaining a cell proliferating state <i>in vivo</i>.

<p>(<b>A</b>) Zebrafish embryos were injected with Myc-tagged <i>deltaA</i> (MT-DeltaA) and <i>d-asb11</i> (Asb11) or <i>asb11^cul</i> (Asb11^cul) mRNA at one-cell stage. (<i>lower panel)</i> 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>B</b>), Fluorescent whole-mount antibody labeling of wild type (WT) and <i>asb11^cul</i> embryos at 24 hpf for the mitotic marker anti-phosphohistone-3 (PH 3) antibody (<i>green</i>) 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.</p

<i>asb11^cul</i> presented altered expression of Delta-Notch pathway components.

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