The localization of Mib during asymmetric division is not known in any experimental system. To address this question in the absence of a working Mib antibody, we used a GFP-tagged full-length Mib (Mib-GFP), which allows examination of the in vivo dynamics of the Mib protein. Multiple-tagged forms of Mib (including GST-, Myc-, and FLAG-tagged versions) have been previously shown to be functional (Itoh et al., 2003). Nevertheless,

we first verified whether the Mib-GFP reflected the endogenous Mib distribution. When transiently expressed in zebrafish embryos through either DNA electroporation or mRNA microinjection, Mib-GFP displayed a punctate pattern that is located in the cytosol near the membrane as well as adjacent to the nucleus (Figures 7A and 7B), in agreement with its previously reported localization and function in endosomes (Itoh et al., 2003 and Koo et al., 2005). In addition we performed double labeling with antibodies Fluorouracil against GFP and Dld at ∼24 hpf. Dld is expressed in the developing brain (Figure 3), albeit less prominently than Dla, for which a workable antibody was not available despite much failed effort with the previously published antibody (Tallafuss et al., 2009). This analysis showed that the Mib-GFP signal was colocalized with Dld (Figure 7C), although find more an exact colocalization was not expected

due to the transient nature of Mib-GFP expression and the presence of other Notch ligands in the brain. Together, these results suggest that Mib-GFP reflects the endogenous Mib distribution pattern. Next, we analyzed the Mib-GFP distribution in paired daughter cells. Coelectroporation of a red fluorescent lineage tracer together with the Mib-GFP construct at ∼22 hpf and analysis of paired daughters at ∼37 hpf showed that Mib-GFP was exclusively

detected in the apical daughter in 85% paired daughter cells analyzed (n = 26) (Figure 7D). This observed percentage is consistent with the idea that Mib asymmetry is likely present in both clone type 1 and 2 (as shown in Figure 1D). In addition the Mib asymmetry appeared to be stably maintained during INM (Figure S6). The unequal segregation of Mib-GFP into the apical daughter made us wonder whether it is dependent on the conserved intrinsic polarity regulator Par-3 because Par-3 has been found asymmetrically over localized to the apical domain of dividing neural progenitors in zebrafish (Alexandre et al., 2010 and von Trotha et al., 2006). We analyzed paired daughters in the embryos injected with a well-established morpholino antisense oligonucleotide targeting par-3 (referred to as the par-3 morphant) ( Alexandre et al., 2010 and Tawk et al., 2007). As expected, the par-3 morphants in our experiments displayed a loss of apicobasal cell polarity and suffered a mild defect in brain morphology at 37 hpf ( Figures S7A–S7I). In the par-3 morphant, Mib-GFP was detected in both daughter cells (91%, n = 23 pairs of daughter cells analyzed) ( Figures 7E and S6).